Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems.  Electroporation  of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells. [ 79 ]  Chemical transfection techniques utilizing  lipids  and peptides have also been used to introduce sgRNAs in complex with Cas9 into cells. [ 80 ] [ 81 ]   Nanoparticle -based delivery has also been used for transfection. [ 82 ]  Types of cells that are more difficult to transfect (e.g., stem cells, neurons, and hematopoietic cells) require more efficient delivery systems, such as those based on  lentivirus  (LVs),  adenovirus  (AdV), and  adeno-associated virus  (AAV). [ 83 ] [ 84 ] [ 85 ]


Efficiency of CRISPR-Cas9 has been found to greatly increase when various components of the system including the entire CRISPR/Cas9 structure to Cas9-gRNA complexes delivered in assembled form rather than using transgenics. [ 86 ] [ 87 ]  This has found particular value in  genetically modified crops  for mass commercialization. [ 88 ] [ 89 ]  Since the host's replication machinery is not needed to produce these proteins, the chance of the recognizing sequence of the sgRNA is almost none, decreasing the chance of off-target effects. [ 82 ]


Further improvements and variants of the CRISPR-Cas9 system have focused on introducing more control into its use. Specifically, the research aimed at improving this system includes improving its specificity, its efficiency, and the granularity of its editing power. Techniques can further be divided and classified by the component of the system they modify. These include using different variants or novel creations of the Cas protein, using an altogether different effector protein, modifying the sgRNA, or using an algorithmic approach to identify existing optimal solutions.


Specificity is an important aspect to improve the CRISPR-Cas9 system because the off-target effects it generates have serious consequences for the genome of the cell and invokes caution for its use. Minimizing off-target effects is thus maximizing the safety of the system. Novel variations of Cas9 proteins that increase specificity include effector proteins with comparable efficiency and specificity to the original SpCas9 that are able to target the previously untargetable sequences and a variant that has virtually no off-target mutations. [ 90 ] [ 91 ]  Research has also been conducted in engineering new Cas9 proteins, including some that partially replace RNA nucleotides in crRNA with DNA and a structure-guided Cas9 mutant generating procedure that all had reduced off-target effects. [ 92 ] [ 93 ]  Iteratively truncated sgRNAs and highly stabilized gRNAs have been shown to also decrease off-target effects. [ 94 ] [ 95 ]  Computational methods including machine learning have been used to predict the affinity of and create unique sequences for the system to maximize specificity for given targets. [ 96 ] [ 97 ]


Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules. [ 98 ] [ 99 ] [ 100 ]  These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation, [ 101 ] [ 102 ]  or by fusing similar light-responsive domains with two constructs of split-Cas9, [ 103 ] [ 104 ]  or by incorporating caged unnatural amino acids into Cas9, [ 105 ]  or by modifying the guide RNAs with photocleavable complements for genome editing. [ 106 ]


Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of  4-hydroxytamoxifen  (4-HT), [ 98 ]  4-HT responsive  intein -linked Cas9, [ 107 ]  or a Cas9 that is 4-HT responsive when fused to four ERT2 domains. [ 108 ]  Intein-inducible split-Cas9 allows  dimerization  of Cas9 fragments [ 109 ]  and  rapamycin -inducible split-Cas9 system developed by fusing two constructs of split-Cas9 with FRB and  FKBP  fragments. [ 110 ]  Other studies have been able to induce transcription of Cas9 with a small molecule,  doxycycline . [ 111 ] [ 112 ]  Small molecules can also be used to improve homology directed repair, [ 113 ]  often by inhibiting the non-homologous end joining pathway and/or the theta-mediated end-joining pathway. [ 114 ] [ 115 ]  A system with the Cpf1 effector protein was created that is induced by small molecules VE-822 and AZD-7762. [ 116 ]  These systems allow conditional control of CRISPR activity for improved precision, efficiency, and spatiotemporal control. Spatiotemporal control is a form of removing off-target effects—only certain cells or parts of the organism may need to be modified, and thus light or small molecules can be used as a way to conduct this. Efficiency of the CRISPR-Cas9 system is also greatly increased by proper delivery of the DNA instructions for creating the proteins and necessary reagents. [ 116 ]


CRISPR also utilizes single base-pair editing proteins to create specific edits at one or two bases in the target sequence. CRISPR/Cas9 was fused with specific enzymes that initially could only change C to T and G to A mutations and their reverse. This was accomplished eventually without requiring any DNA cleavage. [ 117 ] [ 118 ] [ 119 ]  With the fusion of another enzyme, the base editing CRISPR-Cas9 system can also edit C to G and its reverse. [ 120 ]


The clustered regularly interspaced short palindromic repeats  (CRISPR)/Cas9  system is a gene-editing technology that can induce double-strand breaks (DSBs) anywhere guide ribonucleic acids ( gRNA ) can bind with the  protospacer adjacent motif  (PAM) sequence. [ 121 ]  Single-strand nicks can also be induced by Cas9 active-site mutants, [ 122 ]  also known as Cas9 nickases. [ 123 ]  By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs. [ 124 ]  The efficiency of Cas9-endonuclease and the ease by which genes can be targeted led to the development of CRISPR-knockout (KO) libraries both for mouse and human cells, which can cover either specific gene sets of interest or the whole-genome. [ 125 ] [ 126 ]  CRISPR screening helps scientists to create a systematic and high-throughput genetic perturbation within live model organisms. This genetic perturbation is necessary for fully understanding gene function and epigenetic regulation. [ 127 ]  The advantage of pooled CRISPR libraries is that more genes can be targeted at once.


Knock-out libraries are created in a way to achieve equal representation and performance across all expressed gRNAs and carry an antibiotic or fluorescent selection marker that can be used to recover transduced cells. [ 121 ]  There are two  plasmid  systems in CRISPR/Cas9 libraries. First, is all in one plasmid, where sgRNA and Cas9 are produced simultaneously in a transfected cell. Second, is a two-vector system: sgRNA and Cas9 plasmids are delivered separately. [ 127 ]  It is important to deliver thousands of unique sgRNAs-containing vectors to a single vessel of cells by viral transduction at low  multiplicity of infection  (MOI, typically at 0.1–0.6), it prevents the probability that an individual cell clone will get more than one type of sgRNA otherwise it can lead to incorrect assignment of  genotype  to  phenotype . [ 125 ]


Once a pooled library is prepared it is necessary to carry out a deep sequencing (NGS, next generation sequencing) of PCR-amplified plasmid DNA in order to reveal abundance of sgRNAs. Cells of interest can be consequentially infected by the library and then selected according to the phenotype. There are 2 types of selection: negative and positive. By negative selection dead or slow growing cells are efficiently detected. It can identify survival-essential genes, which can further serve as candidates for molecularly targeted drugs. On the other hand, positive selection gives a collection of growth-advantage acquired populations by random mutagenesis. [ 121 ]  After selection genomic DNA is collected and sequenced by NGS. Depletion or enrichment of sgRNAs is detected and compared to the original sgRNA library, annotated with the target gene that sgRNA corresponds to. Statistical analysis then identifies genes that are significantly likely to be relevant to the phenotype of interest. [ 125 ]


113585  TBG backbone


73633  (2 plasmid)


73178  (2 plasmid)


Apart from knock-out there are also knock-down (CRISPRi) and activation (CRISPRa) libraries, which using the ability of proteolytically deactivated Cas9-fusion proteins (dCas9) to bind target DNA, which means that gene of interest is not cut but is over-expressed or repressed. It made CRISPR/Cas9 system even more interesting in gene editing. Inactive dCas9 protein modulate gene expression by targeting dCas9-repressors or activators toward promoter or transcriptional start sites of target genes. For repressing genes Cas9 can be fused to KRAB effector domain that makes complex with gRNA, whereas CRISPRa utilizes dCas9 fused to different transcriptional activation domains, which are further directed by gRNA to promoter regions to upregulate expression. [ 129 ] [ 130 ] [ 131 ]


Cas9 genomic modification has allowed for the quick and efficient generation of  transgenic  models within the field of genetics. Cas9 can be easily introduced into the target cells along with sgRNA via plasmid transfection in order to model the spread of diseases and the cell's response to and defense against infection. [ 132 ]  The ability of Cas9 to be introduced  in vivo  allows for the creation of more accurate models of gene function and mutation effects, all while avoiding the off-target mutations typically observed with older methods of genetic engineering.


The CRISPR and Cas9 revolution in genomic modeling does not extend only to mammals. Traditional genomic models such as  Drosophila melanogaster , one of the first model organisms, have seen further refinement in their resolution with the use of Cas9. [ 132 ]  Cas9 uses cell-specific promoters allowing a controlled use of the Cas9. Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types. The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy. [ 133 ]


CRISPR-Cas9 can be used to edit the DNA of organisms  in vivo  and to eliminate individual genes or even entire  chromosomes  from an organism at any point in its development. Chromosomes that have been successfully deleted  in vivo  using CRISPR techniques include the Y chromosome and X chromosome of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and  aneuploid  mice respectively. This method might be useful for treating genetic disorders caused by abnormal numbers of chromosomes, such as  Down syndrome  and  intersex  disorders. [ 134 ]


Successful  in vivo  genome editing using CRISPR-Cas9 has been shown in numerous model organisms, including  Escherichia coli , [ 135 ]   Saccharomyces cerevisiae , [ 136 ] [ 137 ] Candida albicans , Methanosarcina acetivorans , [ 138 ] [ 139 ]   Caenorhabditis elegans , [ 140 ]   Arabidopsis  spp., [ 141 ]   Danio rerio , [ 142 ]  and  Mus musculus . [ 143 ] [ 144 ]  Successes have been achieved in the study of basic biology, in the creation of disease models, [ 140 ] [ 145 ]  and in the experimental treatment of disease models. [ 146 ]


Concerns have been raised that  off-target effects  (editing of genes besides the ones intended) may confound the results of a CRISPR gene editing experiment (i.e. the observed phenotypic change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. Orthogonal CRISPR experiments are often recommended to confirm the results of a gene editing experiment. [ 147 ] [ 148 ]


CRISPR simplifies the creation of  genetically modified organisms  for research which mimic disease or show what happens when a gene is  knocked down  or mutated. CRISPR may be used at the  germline  level to create organisms in which the targeted gene is changed everywhere (i.e. in all cells/tissues/organs of a multicellular organism), or it may be used in non-germline cells to create local changes that only affect certain cell populations within the organism. [ 149 ] [ 150 ] [ 151 ]


CRISPR can be utilized to create human cellular models of disease. [ 152 ]  For instance, when applied to human  pluripotent stem cells , CRISPR has been used to introduce targeted mutations in genes relevant to  polycystic kidney disease  (PKD) and  focal segmental glomerulosclerosis  (FSGS). [ 153 ]  These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney  organoids  that exhibited disease-specific phenotypes. Kidney  organoids  from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter. [ 154 ]  Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between  podocytes , the filtering cells affected in that disease. This was traced to the inability of podocytes to form microvilli between adjacent cells. [ 155 ]  Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications. [ 153 ]


A similar approach was taken to model  long QT syndrome  in  cardiomyocytes  derived from pluripotent stem cells. [ 156 ]  These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.


CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause. [ 157 ]  Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases, [ 158 ]  including cancer, [ 159 ]   progeria , [ 160 ]  beta-thalassemia, [ 161 ] [ 162 ] [ 163 ]   sickle cell disease , [ 163 ] [ 164 ]   hemophilia , [ 165 ]   cystic fibrosis , [ 166 ]   Duchenne's muscular dystrophy , [ 167 ]   Huntington's disease , [ 168 ] [ 169 ]   transthyretin amyloidosis [ 43 ]  and heart disease. [ 170 ]  CRISPR has also been used to cure  malaria  in mosquitos, which could eliminate the vector and the disease in humans. [ 171 ]  CRISPR may also have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of  MHC class II  proteins, which often cause transplant rejection. [ 172 ]


In addition, clinical trials to cure  beta thalassemia  and  sickle cell disease  in human patients using CRISPR-Cas9 technology have shown promising results. [ 173 ] [ 174 ]  In December 2023, the  US Food and Drug Administration  (FDA) approved the first cell-based gene therapies for treating sickle cell disease,  Casgevy  and  Lyfgenia . Casgevy is the first FDA approved gene therapy to use the CRISPR-Cas9 technology and works by modifying a patient's hematopoietic  stem cells .  [ 175 ]


Nevertheless, there remains a few limitations of the technology's use in  gene therapy : the relatively high frequency of  off-target effect , the requirement for a  PAM sequence  near the target site,  p53  mediated apoptosis by CRISPR-induced  double-strand breaks  and  immunogenic toxicity  due to the delivery system typically by  virus . [ 176 ]


CRISPR has also found many applications in developing cell-based immunotherapies. [ 177 ]  The first clinical trial involving CRISPR started in 2016. It involved taking immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administering the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, as of 2017 [update] . [ 159 ]


In 2016, the  United States Food and Drug Administration  (FDA) approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different kinds of cancer and then administer those engineered T cells back to the same people. [ 178 ]


In November 2020, in mouse animal models, CRISPR was used effectively to treat  glioblastoma  (fast-growing brain tumor) and  metastatic   ovarian cancer , as those are two cancers with some of the worst best-case prognosis and are typically diagnosed during their later stages. The treatments have resulted in inhibited tumor growth, and increased survival by 80% for metastatic ovarian cancer and tumor cell  apoptosis , inhibited tumor growth by 50%, and improved survival by 30% for glioblastoma. [ 179 ]


In October 2021, CRISPR Therapeutics announced results from their ongoing US-based Phase 1 trial for an allogeneic T cell therapy. These cells are sourced from healthy donors and are edited to attack cancer cells and avoid being seen as a threat by the recipient's immune system, and then multiplied into huge batches which can be given to large numbers of recipients. [ 180 ]


In December 2022, a 13-year British girl that had been diagnosed with incurable  T-Cell Acute Lymphoblastic Leukaemia  was cured by doctors at  Great Ormond Street Hospital , in the first documented use of  therapeutic gene editing  for this purpose, after undergoing six months of an experimental treatment, where previous attempts of other treatments failed. The procedure included reprogramming a healthy T-Cell to destroy the cancerous T-Cells to first rid her of Leukaemia, and then rebuilding her immune system from scratch using healthy immune cells. [ 181 ]  The team used  BASE editing  and had  previously  treated a case of  acute lymphoblastic leukaemia  in 2015 using  TALENs . [ 182 ]


Type 1 Diabetes is an endocrine disorder which results from a lack of pancreatic beta cells to produce insulin, a vital compound in transporting blood sugar to cells for producing energy. Researchers have been trying to transplant healthy beta cells. CRISPR is used to edit the cells in order to reduce the chance the patient's body will reject the transplant.


On November 17, 2021 CRISPR therapeutics and ViaCyte announced that the Canadian medical agency had approved their request for a clinical trial for VCTX210, a CRISPR-edited stem cell therapy designed to treat type 1 diabetes. This was significant because it was the first ever gene-edited therapy for diabetes that approached clinics. The same companies also developed a novel treatment for type 1 diabetes to produce insulin via a small medical implant that uses millions of pancreatic cells derived from CRISPR gene-edited stem cells. [ 183 ]


In February 2022, a phase 1 trial was conducted in which one patient volunteer received treatment. [ 180 ] [ 184 ]


Human immunodeficiency virus or HIV, is a virus that attacks the body's immune system. While effective treatments exist which can allow patients to live healthy lives, HIV is retroactive meaning that it embeds an inactive version of itself in the human genome. CRISPR can be used to selectively remove the virus from the genome by designing guide RNA to target the retroactive HIV genome. One issue with this approach is that it requires the removal of the HIV genome from almost all cells, which can be difficult to realistically achieve. [ 180 ]


Initial results in the treatment and cure of HIV have been rather successful, in 2021 9 out of 23 humanized mice treated with a combination of anti-retrovirals and CRISPR/Cas-9 had the virus become undetectable, even after the usual rebound period. None of the two treatments alone had such an effect. [ 185 ]  Clinical trials in humans of a CRISPR–Cas9 based therapy,  EBT-101  started in 2022. [ 186 ] [ 187 ]  In October 2023 an early-stage study on 3 people of EBT-101 reported that the treatment appeared to be safe with no major side effects but no data on its effectiveness was disclosed. [ 188 ]  In March 2024 another CRISPR therapy from researchers of the university of Amsterdam reported the elimination of HIV in cell cultures. [ 189 ] [ 190 ]


CRISPR-Cas-based "RNA-guided nucleases" can be used to target  virulence factors , genes encoding  antibiotic resistance , and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations. [ 191 ] [ 192 ]  Recent studies suggest a correlation between the interfering of the CRISPR-Cas locus and acquisition of antibiotic resistance. [ 193 ]  This system provides protection of bacteria against invading foreign DNA, such as  transposons ,  bacteriophages , and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens. [ 193 ]


Therapies based on  CRISPR–Cas3 gene editing  technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens. [ 194 ]  Cas3 is more destructive than the better known Cas9. [ 195 ] [ 196 ]


Research suggests that CRISPR is an effective way to limit replication of multiple  herpesviruses . It was able to eradicate viral DNA in the case of  Epstein–Barr virus  (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for  immunocompromised  patients of viral invaders, or preventing  cold sore  outbreaks and recurrent eye infections by blocking  HSV-1  reactivation. As of August 2016 [update] , these were awaiting testing. [ 197 ]


CRISPR may revive the concept of  transplanting  animal organs into people.  Retroviruses  present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a particular retroviral DNA sequence from the pig genome in a kidney epithelial cell. [ 198 ]  Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time. [ 199 ]


CRISPR can be used to suppress gain of function mutations and to repair loss of function mutations in neurological disorders. [ 200 ]  The gene editing tool has become a foothold in vivo application for assimilation of molecular pathways.


CRISPR is unique to the development of solving neurological diseases for several reasons. For example, CRISPR allows researchers to quickly generate animal and human cell models, allowing them to study how genes function in a nervous system. By introducing mutations that pertain to various diseases within these cells, researchers can study the effects of the changes on nervous system development, function, and behavior. [ 201 ]  They can uncover the molecular mechanisms that contribute to these disorders, which is essential for developing effective treatments. This is particularly useful in modeling and treating complex neurological disorders such as  Alzheimer's ,  Parkinson's , and  epilepsy  among others.


Alzheimer's Disease (AD) is a  neurodegenerative disease  categorized by neuron loss and an accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain. [ 202 ]  Three pathogenic genes that cause early onset AD in humans have been identified, specifically amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2). [ 202 ]  Over 300 mutations have been detected in these genes, resulting in an increase in total β-amyloid (Aβ), Aβ42/40 ratio, and/or Aβ polymerization.


CRISPR has been used to correct for the  dystrophin  gene, which is responsible for  Duchenne muscular dystrophy , and for the  SCN1A  mutation responsible for the epilepsy disorder  Dravet syndrome . [ 203 ] [ 204 ]  A challenge of using CRISPR for neurological treatment is transferring its components across the  blood-brain barrier . However, recent advancements in nanoparticle delivery systems and viral vectors have shown promise in overcoming this hurdle [ citation needed ] . Looking to the future, the use of CRISPR in neuroscience is expected to increase as technology evolves.


The most commonly occurring worldwide eye diseases are  cataract  and  retinitis pigmentosa  (RP). These are caused by a missense mutation in the alpha chain that leads to permanent blindness. A challenge to the use of CRISPR in eye disease is that retinal tissue in the eye is free from body immune response. Researchers' approach for using CRISPR is to bag the gene coding retinal protein and edit the genome. [ 205 ]


The CRISPR treatment for LCA10 (the most common variant of  Leber congenital amaurosis  which is the leading cause of inherited childhood blindness) modifies the patient's defective photoreceptor gene.


In March 2020, the first patient volunteer in this US-based study, sponsored by  Editas Medicine , was given a low-dose of the treatment to test for safety. In June 2021, enrollment began for a high-dose adult and pediatric cohort of 4 patient volunteers each. Dosing of the new cohorts was expected to be completed by July 2022. [ 180 ]  In November 2022, Editas reported that 20% of the patients treated had significant improvements, but also announced that the resulting target population was too small to support continued independent development. [ 206 ]


CRISPR technology has been shown to work efficiently in the treatment of heart disease. In the case of  familial hypercholesterolemia  (FH), cholesterol deposition in the walls of the artery causes blockage of blood flow. This is caused by mutation in low density lipoprotein cholesterol receptors  (LDLC)  which results in excessive release of cholesterol into the blood. This can be treated by deletion of a base pair in exon 4 of the LDLC receptor. This is a  nonsense mutation .  [ citation needed ]


This disease comes under genetic disorders which are caused by mutation occurring in the structure of  hemoglobin  or due to substitution of different amino acids in globin chains. Due to this, the red blood cells (RBC) cause a string of obstacles such as heart failure, hindrance of blood vessels, defects in growth and optical problems. [ 207 ]  To rehabilitate β-hemoglobinopathies, the patient's multipotent cells are transferred in a mice model to study the rate of gene therapy in ex-vivo which results in expression of mRNA and the gene being rectified. Intriguingly RBC half-life was also increased.


Hemophilia  is a loss of function in blood where clotting factors do not work properly. By using CRISPR-Cas9, a  vector  is inserted into bacteria. [ 208 ]  The vector used is Adenoviral vector which helps in correction of genes.


The application of CRISPR in plants was successfully achieved in the year 2013. CRISPR Cas9 has become an influential appliance in editing genomes in crops. It made a mark in present breeding systems, [ 209 ]


To increase yield of cereal crops, the balance of cytokinin is changed.  Cytokinin oxidase/dehydrogenase  (CKX) is an enzyme that inhibits outgrowth buds in rice, [ 210 ]  so the gene that codes for this enzyme was knocked out to increase yield.


Grains have a high amount of amylose polysaccharide. To decrease the amylose content CRISPR is used to alter the amino acids to reduce saccharide production. Moreover, wheat contains the protein  gluten , to which some people are intolerant, causing  celiac disease . The gene editing tool targets the gluten-coding genes which results in wheat with lower gluten production. [ 211 ]


The biotic stress of plants can be reduced by using CRISPR. The bacterial infections on rice leads to activation of transcription of genes, the products of which are susceptible to disease. By using CRISPR, scientists were able to generate heritable resistance to powdery mildew. [ 212 ]


There are currently about 6000 known genetic disorders, most of which are currently untreatable. The role of CRISPR in gene therapy is to substitute exogenous DNA in place of defective genes. [ 213 ]  Gene therapy has made a huge impact and opened many new possibilities in medical biotechnology.


They are two types of base editings:


Cytidine base editor is a novel therapy in which the cytidine (C) changes to thymidine (T).


Adenine base editor (ABE), [ 214 ]  in this there is a change in base complements from adenine (A) to Guanine (G).


The mutations were directly installed in cellular DNA so that the donor template is not required. The base editings can only edit point mutations. Moreover, they can only fix up to four-point mutations. [ 215 ]  To address this problem, the CRISPR system introduced a new technique known as  Cas9  fusion to increase the scale of genes that can be edited.


Furthermore, the CRISPR Cas9 protein can modulate genes either by activating or silencing based on genes of interest. [ 216 ]  There is a nuclease called dCas9 (endonuclease) used to silence or activate the expression of genes.


The researchers are facing many challenges in gene editing. [ 217 ]  The major hurdles coming in the clinical applications are ethical issues and the transport system to the target site. As the units of CRISPR system taken from bacteria, when they are transferred to host cells it produces an immune response against them. Physical, chemical, viral vectors are used as vehicles to deliver the complex into the host. [ citation needed ]  Due to this many complications are arising such as cell damage that leads to cell death. In the case of viral vectors, the capacity of the virus is small and Cas9 protein is large. So, to overcome these new methods were developed in which smaller strains of Cas9 are taken from bacteria. Finally, a great extent of work is still needed to improve the system.


CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences. [ 220 ]  In 2016, the Cas9 nuclease was used to deplete unwanted nucleotide sequences in next-generation sequencing libraries while requiring only 250 picograms of initial RNA input. [ 221 ]  Beginning in 2017, CRISPR associated nucleases were also used for direct diagnostic testing of nucleic acids, down to single molecule sensitivity. [ 222 ] [ 223 ]  CRISPR diversity is used as an analysis target to discern  phylogeny  and diversity in bacteria, such as in  xanthomonads  by Martins  et al. , 2019. [ 224 ] : 552   Early detections of  plant pathogens  by molecular typing of the pathogen's CRISPRs can be used in agriculture as demonstrated by Shen  et al. , 2020. [ 224 ] : 553


By coupling CRISPR-based diagnostics to additional enzymatic processes, the detection of molecules beyond nucleic acids is possible. One example of a coupled technology is SHERLOCK-based Profiling of IN vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in patient samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices. [ 225 ]  CRISPR-Cas platforms are also being explored for detection [ 226 ] [ 227 ] [ 219 ] [ 228 ] [ 229 ]  and inactivation of  SARS-CoV-2 , the virus that causes  COVID-19 . [ 230 ]  Two different comprehensive diagnostic tests, AIOD-CRISPR and SHERLOCK test have been identified for SARS-CoV-2. [ 231 ]  The SHERLOCK test is based on a fluorescently labelled press reporter RNA which has the ability to identify 10 copies per microliter. [ 232 ]  The AIOD-CRISPR helps with robust and highly sensitive visual detection of the viral nucleic acid. [ 233 ]


CRISPR-Cas9 can be used in investigating and identifying the genetic differences of humans to other apes, especially  of the brain . For example, by reintroducing archaic gene variants into  brain organoids  to show an impact on neurogenesis, [ 234 ]  metaphase length of apical progenitors of the developing neocortex, [ 235 ]  or by knockout of a gene in embryonic stem cells to identify a genetic regulator that via early cell shape transition drives  evolutionary expansion of the human forebrain . [ 236 ] [ 237 ]  One study described a major impact of an archaic gene variant on  neurodevelopment [ 238 ] [ 239 ]  which may be an artefact of a CRISPR side effect, [ 240 ] [ 241 ]  as it could not be replicated in a subsequent study. [ 78 ]


Using "dead" versions of Cas9 ( dCas9 ) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity. [ 198 ]  Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated,  epigenetically  modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human cells have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity. [ 10 ]  Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription. [ 242 ]  Cas9 is an effective way of targeting and silencing specific genes at the DNA level. [ 243 ]  In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called  promoters  that immediately precede the target gene. [ 244 ]


Cas9 was used to carry synthetic  transcription factors  that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter. [ 244 ]


In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit  RNA . The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled CRISPR genes. They considered the fusobacterium  Leptotrichia shahii . It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense. [ 245 ]  C2c2 has later been renamed to Cas13a to fit the standard nomenclature for Cas genes. [ 246 ]


Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses.  HIV  and  poliovirus  are such viruses. Bacteria with Cas13 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing. [ 245 ]


CRISPR-Cas systems can also be employed for editing of  micro-RNA  and  long-noncoding RNA  genes in plants. [ 247 ]


Directing edits to correct mutated sequences was first proposed and demonstrated in 1995. [ 248 ]   This initial work used synthetic RNA antisense oligonucleotides complementary to a pre-mature stop codon mutation in a dystrophin sequence to activate A-to-I editing of the stop codon to a read through codon in a model xenopus cell system. [ 248 ]   While this also led to nearby inadvertent A-to-I transitions, A to I (read as G) transitions can correct all three stop codons, but cannot create a stop codon.  Therefore, the changes led >25% correction of the targeted stop codon with read through to a downstream luciferase reporter sequence.  Follow on work by Rosenthal achieved editing of mutated mRNA sequence in mammalian cell culture by directing an oligonucleotide linked to a cytidine deaminase to correct a mutated cystic fibrosis sequence. [ 249 ]   More recently, CRISPR-Cas13 fused to deaminases has been employed to direct mRNA editing. [ 250 ]


Gene drives may provide a powerful tool to restore balance of ecosystems by eliminating invasive species. Concerns regarding efficacy, unintended consequences in the target species as well as non-target species have been raised particularly in the potential for accidental release from laboratories into the wild. Scientists have proposed several safeguards for ensuring the containment of experimental gene drives including molecular, reproductive, and ecological. [ 255 ]  Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary. [ 256 ]  There remains consensus that long-term effects must be studied more thoroughly particularly in the potential for ecological disruption that cannot be corrected with reversal drives. [ 257 ]


Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant  off-target effects  as seen with  restriction enzymes . Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries. [ 258 ]


Epigenome editing  or epigenome engineering is a type of genetic engineering in which the  epigenome  is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.


Targeted regulation of disease-related genes may enable novel therapies for many diseases, especially in cases where adequate gene therapies are not yet developed or are inappropriate. [ 259 ]  While transgenerational and population level consequences are not fully understood, it may become a major tool for applied functional genomics and  personalized medicine . [ 260 ]  As with  RNA editing , it does not involve genetic changes and their accompanying risks. [ 259 ]  One example of a potential functional use of epigenome editing was described in 2021: repressing  Na v 1.7   gene expression  via  CRISPR-dCas9  which showed therapeutic potential in three mouse models of chronic pain. [ 261 ] [ 262 ]


Combination of CRISPR-Cas9 with  integrases  enabled a technique for  large edits  without problematic double-stranded breaks, as demonstrated with  PASTE  in 2022. The researchers reported it could be used to deliver genes as long as 36,000 DNA  base pairs  to several types of human cells and thereby potentially for treating diseases caused by a large number of mutations. [ 264 ] [ 265 ]


Prime editing [ 266 ]  (or base editing) is a CRISPR refinement to accurately insert or delete sections of DNA. The CRISPR edits are not always perfect and the cuts can end up in the wrong place. Both issues are a problem for using the technology in medicine. [ 267 ]  Prime editing does not cut the double-stranded DNA but instead uses the CRISPR targeting apparatus to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another. [ 268 ]  The new guide, called a pegRNA, contains an RNA template for a new DNA sequence to be added to the genome at the target location. That requires a second protein, attached to Cas9: a reverse transcriptase enzyme, which can make a new DNA strand from the RNA template and insert it at the nicked site. [ 269 ]  Those three independent pairing events each provide an opportunity to prevent off-target sequences, which significantly increases targeting flexibility and editing precision. [ 268 ]  Prime editing was developed by researchers at the  Broad Institute of MIT and Harvard  in Massachusetts. [ 270 ]  More work is needed to optimize the methods. [ 270 ] [ 269 ]


As of March 2015, multiple groups had announced ongoing research with the intention of laying the foundations for applying CRISPR to human embryos for  human germline engineering , including labs in the US, China, and the UK, as well as US biotechnology company  OvaScience . [ 271 ]  Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations". [ 272 ] [ 273 ]  These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans. [ 274 ]


In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable  human embryos  using CRISPR to correct a mutation that causes  beta thalassemia , a lethal heritable disorder. [ 275 ] [ 276 ]  The study had previously been rejected by both  Nature  and  Science  in part because of ethical concerns. [ 277 ]  The experiments resulted in successfully changing only some of the intended genes, and had  off-target effects  on other genes. The researchers stated that CRISPR is not ready for clinical application in  reproductive medicine . [ 277 ]  In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR – this time to alter the  CCR5  gene to make the embryo resistant to  HIV  infection. [ 278 ]


In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of  David Baltimore . Members of national scientific academies of the US, UK, and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between  somatic cells , where the effects of edits are limited to a single individual, and germline cells, where genome changes can be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene–environment interactions) and culturally (e.g.  social Darwinism ). Altering of  gametocytes  and embryos to generate heritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries. [ 279 ]


In February 2017, the  United States National Academies of Sciences, Engineering, and Medicine  ( NASEM ) Committee on Human Gene Editing published a report reviewing ethical, legal, and scientific concerns of genomic engineering technology. The conclusion of the report stated that heritable  genome editing  is impermissible now but could be justified for certain medical conditions; however, they did not justify the usage of  CRISPR  for enhancement. [ 280 ]


In November 2018,  Jiankui He  announced that he had edited two human embryos to attempt to disable the gene for  CCR5 , which codes for a receptor that  HIV  uses to enter cells. He said that twin girls,  Lulu and Nana , had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 ( mosaicism ) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature. [ 281 ]  An international group of scientists called for a global moratorium on genetically editing human embryos. [ 282 ]


Designer babies


The advent of CRISPR-Cas9 gene editing technology has led to the possibility of creating "designer babies." This technology has the possibility of eliminating certain genetic diseases, or improving health by enhancing certain genetic traits.


Policy regulations for the CRISPR-Cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify  human embryos  by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days. [ 283 ]


The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the  Agriculture Risk Protection Act of 2000  gives the  United States Department of Agriculture  the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment, and economy of the US. The act regulates any  genetically modified organism  that utilizes the genome of a predefined "plant pest" or any plant not previously categorized. [ 284 ]  In 2015, Yinong Yang successfully deactivated 16 specific genes in the  white button mushroom  to make them non-browning. Since he had not added any foreign-species ( transgenic ) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2. [ 285 ]  Yang's white button mushroom was the first organism genetically modified with the CRISPR-Cas9 protein system to pass US regulation. [ 286 ]


In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US  National Academies of Sciences, Engineering, and Medicine , special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result. [ 287 ]  In 2017, the  Food and Drug Administration  proposed a rule that would classify genetic engineering modifications to animals as "animal drugs", subjecting them to strict regulation if offered for sale and reducing the ability for individuals and small businesses to make them profitable. [ 288 ] [ 289 ]


In China, where social conditions sharply contrast with those of the West, genetic diseases carry a heavy stigma. [ 290 ]  This leaves China with fewer policy barriers to the use of this technology. [ 291 ] [ 292 ]


In 2012 and 2013, CRISPR was a runner-up in  Science Magazine 's  Breakthrough of the Year  award. In 2015, it was the winner of that award. [ 198 ]  CRISPR was named as one of  MIT Technology Review ' s 10 breakthrough technologies in 2014 and 2016. [ 293 ] [ 294 ]  In 2016,  Jennifer Doudna  and  Emmanuelle Charpentier , along with Rudolph Barrangou,  Philippe Horvath , and  Feng Zhang  won the Gairdner International award. In 2017, Doudna and Charpentier were awarded the Japan Prize in Tokyo, Japan for their revolutionary invention of CRISPR-Cas9. In 2016, Charpentier, Doudna, and Zhang won the  Tang Prize  in Biopharmaceutical Science. [ 295 ]  In 2020, Charpentier and Doudna were awarded the  Nobel Prize in Chemistry , the first such prize for an all-female team, "for the development of a method for genome editing." [ 296 ]


CRISPR-Display ( CRISP-Disp )  is a modification of the  CRISPR/Cas9  (Clustered regularly interspaced short palindromic repeats) system for genome editing. The CRISPR/Cas9 system uses a short guide RNA (sgRNA) sequence to direct a  Streptococcus pyogenes   Cas9  nuclease, acting as a programmable DNA binding protein, to cleave DNA at a site of interest. [ 1 ] [ 2 ]


CRISPR-Display, in contrast, uses a nuclease deficient Cas9 (dCas9) and an engineered sgRNA with aptameric accessory RNA domains, ranging from 100bp to 5kb, outside of the normal complementary targeting sequence. [ 3 ]  The accessory RNA domains can be functional domains, such as long non-coding RNAs ( lncRNAs ), protein-binding motifs, or epitope tags for immunochemistry. This allows for investigation of the functionality of certain lncRNAs, and targeting of  ribonucleoprotein  (RNP) complexes to genomic loci.


CRISPR-Display was first published in Nature Methods in July 2015, and developed by David M. Shechner, Ezgi Hacisuleyman, Scott T. Younger and John Rinn at  Harvard University  and  Massachusetts Institute of Technology  (MIT), USA.


The CRISPR/Cas9 system is based on an adaptive immune system of prokaryotic organisms, and its use for genome editing was first proposed and developed in collaboration between  Jennifer Doudna  ( University of California, Berkeley ) and  Emmanuelle Charpentier  ( Max Planck Institute for Infection Biology , Germany). [ 1 ]  The method, and its application in editing human cells, was published in Science on August 17, 2012. In January 2013, the  Feng Zhang  lab at the  Broad Institute  at MIT published another method in Science, having further optimized the sgRNA structure and expression for use in mammalian cells. [ 2 ]  By the beginning of 2014, almost 2500 studies mentioning CRISPR in their title has been published.


Non-coding RNAs  (ncRNAs) are RNA transcripts that are not translated into a protein product, but instead exert their function as RNA molecules. They are involved in a range of processes, like  post-transcriptional regulation  of gene expression,  genomic imprinting , and regulating the chromatin state, and thereby the expression, of a given locus. Many ncRNAs have been discovered, but in many cases, their function has yet to be accurately dissected due to technical challenges. ncRNA function is often not affected by introducing point mutations and premature stop codons. ncRNAs are also thought to regulate gene expression, so deletion studies have a hard time distinguishing effects of ncRNA loss from effects of gene misregulation due to the deletion. Studies of ncRNAs have also lacked the throughput necessary for discerning the RNA based functionality. To meet these challenges, the Rinn lab therefore developed a synthetic biology approach, using CRISPR/Cas9 system, with the Cas9 acting as a conduit, to target ncRNA modules to ectopic genomic locations, and investigating the ncRNAs effects on reporter genes and other genomic features at that site.


CRISPR-Disp modifies the CRISPR/Cas9 technology by using a catalytically inactive, i.e. nuclease deficient, Cas9 mutant (dCas9), and altering the RNA used for targeting Cas9 to a genomic location.


Since sgRNAs are usually expressed by  RNA polymerase III , which limits the length of the RNA domain that can be inserted, CRISPR-Display incorporates  RNA polymerase II  to permit expression of longer transcripts (~80–250 nucleotides) to overcome this limitation. CRISPR-Display can therefore add larger RNA domains, like natural and lncRNA domains, without affecting dCas9 localization.


The sgRNA is engineered with an aptameric accessory RNA domain in the sequence outside of the targeting sequence. In the development of the technique, five model cofactors with different topology constructs were used: TOP1-4 and INT with an accessory domain (P4-P6 domain) at different positions, including the 5’ and 3’ end and internally within the sgRNA. Each domain contained a stem-loop that can be recognized by a PP7 bacteriophage coat protein. The complex was delivered into mammalian cells (HEK293FT cells) by a  lentiviral vector .


To ensure that the attached RNA module both retains targeting functionality as well as the resulting complex drive transcriptional activation at a specific site of interest, transient reporter gene expression of  luciferase  and fluorescent protein was measured. Two variations of such a  transcription activator assay  was performed; directly with a dCas9 fused to a transcriptional activator/repressor (VP64, a factor known to enhance gene expression)  (Direct activation)  or indirectly where the transcriptional activator is fused to an RNA binding protein module on the sgRNA  (Bridged activation) . Reporter gene activation through direct activation imply the sgRNA variant binds and targets dCas9 efficiently. All the five topologies showed direct activation except TOP3 and TOP4, which showed reduced activity. Bridged activation indicates that the fused RNA accessory domain is intact in mature dCas9 complexes. Bridged activation was observed with TOP1, TOP3 and INT. The results were recapitulated at endogenous loci by targeting minimal sgRNA and selective expanded topologies (TOP1 and INT) to human ASCL1, IL1RN, NTF3 and TTN promoters. Direct and bridged activation were observed by qRT-PCR for each construct proving that CRISP-Disp allows deployment of large RNA domains to genomic loci.


The effect of internal (stem-loop) insertion size on dCas9 complex was assessed using INT-like constructs with cassettes of PP7 stem loops with a size range from 25 nt to 247 nt. Each construct induced significant activation in the reporter assays signifying that internal insertion size does not influence the dCas9 complex function. Similarly, the effect of internal insert sequence was also determined through a set of unique sgRNA variants displaying cassettes of 25 random nucleotides. Reporter assays and RIP-Seq confirmed that sequence does not govern complex efficacy.


The utility of CRISP-Disp was explored with an array of functional RNA domains such as natural protein binding motifs, artificial aptamers and small molecules with varying size. While all the complexes were functional and viable, and successfully deployed the RNA domains at endogenous loci, the efficacy changed with length and expression levels. This suggests that optimization of structure and sequence might be important required before designing the construct.


To determine if artificial lncRNA scaffolds can be used with CRISPR-Display, dCas9 complexes were assembled with artificial RNA with a size comparable to lncRNAs. The constructs were expanded to ~650nt size with an additional P4-P6 domain with hairpin loops that can be recognized by another phage coat protein, MS2. These topology constructs were called double TOP0-2 with the two domains either together at 5’ or 3’end or separately at each end. Transient reporter assays followed by confirmation with  RNA Immunoprecipitation  sequencing (RIP-qPCR) showed that all the three constructs retained both the domains in the complex.


This was also tested with natural lncRNA domains by building Pol II-driven TOP1 and INT constructs fused with human lncRNA domains. lncRNAs used had lengths between ~90–4800 nt, and included the NoRC-binding pRNA, three enhancer-transcribed RNAs (eRNAs) FALEC, TRERNA1 and ncRNA-a3),  Xist  A-repeat (RepA), and the 4,799-nt transcriptional activator  HOTTIP . While all the constructs showed significant direct activation, it decreased with increasing lncRNA-sgRNA length. These lncRNA domains could regulate the reporters independent of dCas9 with pRNA and RepA repressing the GLuc reporter expression (repressors) and TRERNA1, ncRNA-a3 and HOTTIP inducing activation (activators), but were properly targeted to an ectopic location of interest by using the CRISP-Disp system. [ 3 ]


Thus, CRISP-Disp enables control of gene expression with deployment of both artificial scaffolds as well as natural lncRNA domains.


CRISPR-Display allows for previously unavailable studies of lncRNA functionality, artificial ncRNA functionalization, recruitment of endogenous and engineered proteins to genomic loci, and locus affinity tagging for cell imaging.


CRISPR-Display allows targeted localization of natural lncRNAs to ectopic sites for investigation of their function. Exposing various ectopic DNA loci to natural lncRNAs can help show the effects of lncRNAs on gene expression and chromatin state, and help dissect the mechanism of such effects. One of the major outstanding questions in the study of lncRNAs is whether effects on chromatin state or gene expression adjacent to a lncRNA locus is due to functional, sequence-specific mechanisms of the lncRNA itself, or due simply to the act of transcribing the lncRNA. [ 4 ] [ 5 ]  Localizing lncRNA to ectopic sites with CRISPR-Display can help separate the function of the RNA itself from the effects of transcribing such RNA species. Before CRISPR-Display, such studies were challenging due to low throughput, and inability to distinguish lncRNA function from other confounding factors like cryptically encoded peptides or functional DNA elements.


CRISPR-Display also allows for targeted use of the wide array of artificial RNAs with specific functionality, such as RNAs for recruitment of endogenous RNA-binding proteins, antibody affinity tagging, and recruitment of tagged fusion proteins.


One example of artificial ncRNA functionalization is incorporating RNA domains recognized by specific antibodies to the sgRNA. CRISPR-Display can target the sgRNA with a particular epitope sequence to various loci, and fluorescently tagged antibodies can be used to image the locus, showing its localization in the nucleus, and possible interactions with other tagged proteins or genomic loci.


Endogenous proteins known to bind a specific RNA motif can be recruited to ectopic genomic locations by incorporating the RNA motif into the sgRNA. CRISPR-Display can also recruit fusion proteins engineered to bind specific RNA sequences. Recruiting these proteins can allow studies of specific proteins’ and protein complexes’ effects on gene regulation and chromatin states, as well as specific regulation of certain genes for investigation of gene function.


Due to the modularity of the sgRNA, several different sgRNAs with distinct functional modules can be expressed in each cell at once. The different RNA modules can then work simultaneously and independently, allowing for, for example, regulation of one genomic location whilst imaging the effects of the regulation at another location.


The possible applications of CRISPR-Display will continue to increase with further development and understanding of ncRNA functionalization. It is not unreasonable to think that CRISPR-Display may one day enable complex synthetic biology systems, with distinct temporal expression of sgRNAs, and networks and circuits of gene regulation by targeting of regulatory proteins.


A  designer baby  is a baby whose  genetic makeup  has been selected or altered, often to exclude a particular  gene  or to remove genes associated with disease. [ 1 ] [ 2 ]  This process usually involves analysing a wide range of human  embryos  to identify genes associated with particular  diseases  and characteristics, and selecting embryos that have the desired genetic makeup; a process known as  preimplantation genetic diagnosis . [ 3 ]  Screening for single genes is commonly practiced, and polygenic screening is offered by a few companies. [ 4 ]  Other methods by which a baby's genetic information can be altered involve directly  editing the genome  before birth, which is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins  Lulu and Nana  were edited as embryos, causing widespread criticism. [ 5 ]


Genetically altered embryos can be achieved by introducing the desired genetic material into the embryo itself, or into the  sperm  and/or  egg  cells of the parents; either by  delivering the desired genes directly into the cell  or using gene-editing technology. This process is known as  germline engineering  and performing this on embryos that will be brought to term is typically prohibited by law. [ 6 ]  Editing embryos in this manner means that the genetic changes can be  carried down to future generations , and since the technology concerns editing the genes of an unborn baby, it is considered controversial and is subject to ethical debate. [ 7 ]  While some scientists condone the use of this technology to treat disease, concerns have been raised that this could be translated into using the technology for cosmetic purposes and  enhancement of human traits . [ 8 ]


Pre-implantation genetic diagnosis (PGD or PIGD) is a procedure in which embryos are screened prior to  implantation . The technique is used alongside  in vitro  fertilisation  (IVF) to obtain embryos for evaluation of the genome – alternatively, ovocytes can be screened prior to  fertilisation . The technique was first used in 1989. [ 9 ]


PGD is used primarily to select embryos for implantation in the case of possible  genetic defects , allowing identification of  mutated  or disease-related  alleles  and selection against them. It is especially useful in embryos from parents where one or both carry a  heritable disease . PGD can also be used to select for embryos of a certain sex, most commonly when a disease is more strongly associated with one sex than the other (as is the case for  X-linked disorders  which are more common in males, such as  haemophilia ). Infants born with traits selected following PGD are sometimes considered to be designer babies. [ 10 ]


One application of PGD is the selection of ' saviour siblings ', children who are born to provide a  transplant  (of an organ or group of cells) to a sibling with a usually life-threatening disease. Saviour siblings are conceived through IVF and then screened using PGD to analyze genetic similarity to the child needing a transplant, to reduce the risk of  rejection . [ 11 ]


Embryos for PGD are obtained from IVF procedures in which the oocyte is artificially fertilised by sperm.  Oocytes  from the woman are harvested following  controlled ovarian hyperstimulation  (COH), which involves fertility treatments to induce production of multiple oocytes. After harvesting the oocytes, they are fertilised  in vitro , either during incubation with multiple sperm cells in culture, or  via   intracytoplasmic sperm injection  (ICSI), where sperm is directly injected into the oocyte. The resulting embryos are usually cultured for 3–6 days, allowing them to reach the  blastomere  or  blastocyst  stage. [ 12 ]


Once embryos reach the desired stage of development, cells are biopsied and genetically screened. The screening procedure varies based on the nature of the disorder being investigated.


Polymerase chain reaction  (PCR) is a process in which  DNA  sequences are amplified to produce many more copies of the same segment, allowing screening of large samples and identification of specific genes. [ 13 ]  The process is often used when screening for  monogenic disorders , such as  cystic fibrosis .


Another screening technique,  fluorescent  in situ  hybridisation  (FISH) uses fluorescent probes which specifically bind to highly  complementary sequences  on  chromosomes , which can then be identified using  fluorescence microscopy . [ 14 ]  FISH is often used when screening for chromosomal abnormalities such as  aneuploidy , making it a useful tool when screening for disorders such as  Down syndrome .


Following the screening, embryos with the desired trait (or lacking an undesired trait such as a mutation) are transferred into the mother's  uterus , then allowed to  develop naturally .


PGD regulation is determined by individual countries' governments, with some prohibiting its use entirely, including in  Austria ,  China , and  Ireland . [ 15 ]


In many countries, PGD is permitted under very stringent conditions for medical use only, as is the case in  France ,  Switzerland ,  Italy  and the  United Kingdom . [ 16 ] [ 17 ]  Whilst PGD in Italy and Switzerland is only permitted under certain circumstances, there is no clear set of specifications under which PGD can be carried out, and selection of embryos based on sex is not permitted. In France and the UK, regulations are much more detailed, with dedicated agencies setting out framework for PGD. [ 18 ] [ 19 ]  Selection based on sex is permitted under certain circumstances, and genetic disorders for which PGD is permitted are detailed by the countries' respective agencies.


In contrast, the  United States  federal law does not regulate PGD, with no dedicated agencies specifying regulatory framework by which healthcare professionals must abide. [ 16 ]  Elective sex selection is permitted, accounting for around 9% of all PGD cases in the U.S., as is selection for desired conditions such as  deafness  or  dwarfism . [ 20 ]


Based on the specific analysis conducted:


PGT-M (Preimplantation Genetic Testing for monogenic diseases) : It is used to detect hereditary diseases caused by the  mutation  or alteration of the DNA sequence of a single gene. [ 21 ]


PGT-A (Preimplantation Genetic Testing for aneuploidy) : It is used to diagnose numerical abnormalities ( aneuploidies ). [ 22 ]


Human germline engineering is a process in which the human genome is edited within a  germ cell , such as a sperm cell or oocyte (causing heritable changes), or in the  zygote  or embryo following fertilization. [ 23 ]  Germline engineering results in changes in the genome being incorporated into every cell in the body of the offspring (or of the individual following embryonic germline engineering). This process differs from  somatic cell  engineering, which does not result in heritable changes. Most human germline editing is performed on individual cells and non-viable embryos, which are destroyed at a very early stage of development. In November 2018, however, a Chinese scientist,  He Jiankui , announced that he had created the first human germline genetically edited babies. [ 24 ]


Genetic engineering relies on a knowledge of human genetic information, made possible by research such as the  Human Genome Project , which identified the position and function of all the genes in the human genome. [ 25 ]  As of 2019,  high-throughput sequencing  methods allow  genome sequencing  to be conducted very rapidly, making the technology widely available to researchers. [ 26 ]


Germline modification is typically accomplished through techniques which incorporate a new gene into the genome of the embryo or germ cell in a specific location. This can be achieved by introducing the desired DNA directly to the cell for it to be incorporated, or by replacing a gene with one of interest. These techniques can also be used to remove or disrupt unwanted genes, such as ones containing mutated sequences.


Whilst germline engineering has mostly been performed in  mammals  and other animals, research on human cells  in vitro  is becoming more common. Most commonly used in human cells are  germline gene therapy  and the engineered nuclease system  CRISPR/Cas9 .


Gene therapy  is the delivery of a  nucleic acid  (usually DNA or  RNA ) into a cell as a pharmaceutical agent to treat disease. [ 27 ]  Most commonly it is carried out using a  vector , which transports the nucleic acid (usually DNA encoding a therapeutic gene) into the target cell. A vector can  transduce  a desired copy of a gene into a specific location to be  expressed  as required. Alternatively, a  transgene  can be inserted to deliberately disrupt an unwanted or mutated gene, preventing  transcription  and  translation  of the faulty gene products to avoid a disease  phenotype .


Gene therapy in patients is typically carried out on somatic cells in order to treat conditions such as some  leukaemias  and  vascular diseases . [ 28 ] [ 29 ] [ 30 ]
Human germline gene therapy in contrast is restricted to  in vitro  experiments in some countries, whilst others prohibited it entirely, including  Australia ,  Canada ,  Germany  and Switzerland. [ 31 ]


Whilst the  National Institutes of Health  in the US does not currently allow  in utero  germline gene transfer clinical trials,  in vitro  trials are permitted. [ 32 ]  The NIH guidelines state that further studies are required regarding the safety of gene transfer protocols before  in utero  research is considered, requiring current studies to provide demonstrable efficacy of the techniques in the laboratory. [ 33 ]  Research of this sort is currently using non-viable embryos to investigate the efficacy of germline gene therapy in treatment of disorders such as inherited  mitochondrial diseases . [ 34 ]


Gene transfer to cells is usually by vector delivery. Vectors are typically divided into two classes –  viral  and  non-viral .


Viruses  infect cells by transducing their genetic material into a host's cell, using the host's cellular machinery to generate viral proteins needed for replication and proliferation. By modifying viruses and loading them with the therapeutic DNA or RNA of interest, it is possible to use these as a vector to provide delivery of the desired gene into the cell. [ 35 ]


Retroviruses  are some of the most commonly used viral vectors, as they not only introduce their genetic material into the host cell, but also copy it into the host's genome. In the context of gene therapy, this allows permanent integration of the gene of interest into the patient's own DNA, providing longer lasting effects. [ 36 ]


Viral vectors work efficiently and are mostly safe but present with some complications, contributing to the stringency of regulation on gene therapy. Despite partial inactivation of viral vectors in gene therapy research, they can still be  immunogenic  and elicit an  immune response . This can impede viral delivery of the gene of interest, as well as cause complications for the patient themselves when used clinically, especially in those who already have a serious genetic illness. [ 37 ]  Another difficulty is the possibility that some viruses will randomly integrate their nucleic acids into the genome, which can interrupt gene function and generate new mutations. [ 38 ]
This is a significant concern when considering germline gene therapy, due to the potential to generate new mutations in the embryo or offspring.


Non-viral methods of nucleic acid  transfection  involved injecting a naked DNA  plasmid  into cell for incorporation into the genome. [ 39 ]  This method used to be relatively ineffective with low frequency of integration, however, efficiency has since greatly improved, using methods to enhance the delivery of the gene of interest into cells. Furthermore, non-viral vectors are simple to produce on a large scale and are not highly immunogenic.


Some non-viral methods are detailed below:


Zinc-finger nucleases  (ZFNs) are enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger recognizes between 9 and 18 bases of sequence. Thus by mixing those modules, it becomes easier to target any sequence researchers wish to alter ideally within complex genomes. A ZFN is a  macromolecular  complex formed by monomers in which each subunit contains a zinc domain and a  FokI endonuclease domain.  The FokI domains must dimerize for activities, thus narrowing target area by ensuring that two close DNA-binding events occurs. [ 44 ]


The resulting cleavage event enables most genome-editing technologies to work. After a break is created, the cell seeks to repair it.


The success of using ZFNs in gene therapy depends on the insertion of genes to the chromosomal target area without causing damage to the cell. Custom ZFNs offer an option in human cells for gene correction.


There is a method called  TALENs  that targets singular nucleotides. TALENs stand for transcription activator-like effector nucleases. TALENs are made by  TAL effector DNA-binding domain  to a DNA cleavage domain. All these methods work by as the TALENs are arranged. TALENs are "built from arrays of 33-35 amino acid modules…by assembling those arrays…researchers can target any sequence they like". [ 44 ]  This event is referred as Repeat Variable Diresidue (RVD). The relationship between the amino acids enables researchers to engineer a specific DNA domain. The TALEN enzymes are designed to remove specific parts of the DNA strands and replace the section; which enables edits to be made. TALENs can be used to edit genomes using  non-homologous end joining  (NHEJ) and  homology directed repair .


The CRISPR/Cas9 system ( CRISPR  – Clustered Regularly Interspaced Short Palindromic Repeats,  Cas9  – CRISPR-associated protein 9) is a genome editing technology based on the  bacterial  antiviral CRISPR/Cas system. The bacterial system has evolved to recognize viral nucleic acid sequences and cut these sequences upon recognition, damaging infecting viruses. The gene editing technology uses a simplified version of this process, manipulating the components of the bacterial system to allow location-specific gene editing. [ 45 ]


The CRISPR/Cas9 system broadly consists of two major components – the Cas9  nuclease  and a  guide RNA  (gRNA). The gRNA contains a Cas-binding sequence and a ~20  nucleotide  spacer sequence, which is specific and complementary to the target sequence on the DNA of interest. Editing specificity can therefore be changed by modifying this spacer sequence. [ 45 ]


Upon system delivery to a cell, Cas9 and the gRNA bind, forming a  ribonucleoprotein  complex. This causes a  conformational change  in Cas9, allowing it to cleave DNA if the gRNA spacer sequence binds with sufficient  homology  to a particular sequence in the host genome. [ 46 ]  When the gRNA binds to the target sequence, Cas will cleave the  locus , causing a  double-strand break  (DSB).


The resulting DSB can be  repaired  by one of two mechanisms –


Since NHEJ is more efficient than HDR, most DSBs will be repaired  via  NHEJ, introducing gene knockouts. To increase frequency of HDR, inhibiting genes associated with NHEJ and performing the process in particular  cell cycle  phases (primarily  S  and  G2 ) appear effective.


CRISPR/Cas9 is an effective way of manipulating the genome  in vivo  in animals as well as in human cells  in vitro , but some issues with the efficiency of delivery and editing mean that it is not considered safe for use in viable human embryos or the body's germ cells. As well as the higher efficiency of NHEJ making inadvertent knockouts likely, CRISPR can introduce DSBs to unintended parts of the genome, called off-target effects. [ 47 ]  These arise due to the spacer sequence of the gRNA conferring sufficient sequence homology to random loci in the  genome , which can introduce random mutations throughout. If performed in germline cells, mutations could be introduced to all the cells of a developing embryo.


There are developments to prevent unintended consequences otherwise known as off-target effects due to gene editing. [ 48 ]  There is a race to develop new gene editing technologies that prevent off-target effects from occurring with some of the technologies being known as biased off-target detection, and Anti-CRISPR Proteins. [ 48 ]  For biased off-target effects detection, there are several tools to predict the locations where off-target effects may take place. [ 48 ]  Within the technology of biased off-target effects detection, there are two main models, Alignment Based Models that involve having the sequences of gRNA being aligned with sequences of genome, after which then the off-target locations are predicted. [ 48 ]  The second model is known as the Scoring-Based Model where each piece of gRNA is scored for their off-target effects in accordance with their positioning. [ 48 ]


In 2015, the International Summit on Human Gene Editing was held in  Washington D.C. , hosted by scientists from China, the UK and the U.S. The summit concluded that genome editing of somatic cells using CRISPR and other genome editing tools would be allowed to proceed under  FDA  regulations, but human germline engineering would not be pursued. [ 32 ]


In February 2016, scientists at the  Francis Crick Institute  in  London  were given a license permitting them to edit human embryos using CRISPR to investigate early development. [ 49 ]  Regulations were imposed to prevent the researchers from implanting the embryos and to ensure experiments were stopped and embryos destroyed after seven days.


In November 2018, Chinese scientist  He Jiankui  announced that he had performed the first germline engineering on viable human embryos, which have since been brought to term. [ 24 ]  The research claims received significant criticism, and Chinese authorities suspended He's research activity. [ 50 ]  Following the event, scientists and government bodies have called for more stringent regulations to be imposed on the use of CRISPR technology in embryos, with some calling for a global  moratorium  on germline genetic engineering. Chinese authorities have announced stricter controls will be imposed, with  Communist Party  general secretary  Xi Jinping  and  government  premier  Li Keqiang  calling for new gene-editing legislations to be introduced. [ 51 ] [ 52 ]


As of January 2020, germline genetic alterations are prohibited in 24 countries by law and also in 9 other countries by their guidelines. [ 53 ]  The  Council of Europe 's Convention on Human Rights and Biomedicine, also known as the Oviedo Convention, has stated in its article 13 "Interventions on the human genome" as follows: "An intervention seeking to modify the human genome may only be undertaken for preventive, diagnostic or therapeutic purposes and only if its aim is not to introduce any modification in the genome of any descendants". [ 54 ] [ 55 ]  Nonetheless, wide public debate has emerged, targeting the fact that the Oviedo Convention Article 13 should be revisited and renewed, especially due to the fact that it was constructed in 1997 and may be out of date, given recent technological advancements in the field of genetic engineering. [ 56 ]


The Lulu and Nana controversy refers to the two Chinese twin girls born in November 2018, who had been genetically modified as embryos by the Chinese scientist He Jiankui. [ 24 ]  The twins are believed to be the first genetically modified babies. The girls' parents had participated in a clinical project run by He, which involved IVF, PGD and genome editing procedures in an attempt to edit the gene  CCR5 . CCR5 encodes a protein used by  HIV  to enter host cells, so by introducing a specific mutation into the gene  CCR5 Δ32  He claimed that the process would confer  innate resistance to HIV . [ 57 ] [ 58 ]


The project run by He recruited couples wanting children where the man was  HIV-positive  and the woman uninfected. During the project, He performed IVF with sperm and eggs from the couples and then introduced the CCR5 Δ32 mutation into the genomes of the embryos using CRISPR/Cas9. He then used PGD on the edited embryos during which he sequenced biopsied cells to identify whether the mutation had been successfully introduced. He reported some  mosaicism  in the embryos, whereby the mutation had integrated into some cells but not all, suggesting the offspring would not be entirely protected against HIV. [ 59 ]  He claimed that during the PGD and throughout the pregnancy,  fetal DNA  was sequenced to check for off-target errors introduced by the CRISPR/Cas9 technology, however the NIH released a statement in which they announced "the possibility of damaging off-target effects has not been satisfactorily explored". [ 60 ] [ 61 ]  The girls were born in early November 2018, and were reported by He to be healthy. [ 59 ]


His research was conducted in secret until November 2018, when documents were posted on the Chinese clinical trials registry and  MIT Technology Review  published a story about the project. [ 62 ]  Following this, He was interviewed by the  Associated Press  and presented his work on 27 November at the Second International Human Genome Editing Summit which was held in  Hong Kong . [ 57 ]


Although the information available about this experiment is relatively limited, it is deemed that the scientist erred against many ethical, social and moral rules but also China's guidelines and regulations, which prohibited germ-line genetic modifications in human embryos, while conducting this trial. [ 63 ] [ 64 ]  From a technological point of view, the CRISPR/Cas9 technique is one of the most precise and least expensive methods of gene modification to this day, whereas there are still a number of limitations that keep the technique from being labelled as safe and efficient. [ 64 ]  During the First International Summit on Human Gene Editing in 2015 the participants agreed that a halt must be set on germline genetic alterations in clinical settings unless and until: "(1) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (2) there is broad societal consensus about the appropriateness of the proposed application". [ 64 ]  However, during the second International Summit in 2018 the topic was once again brought up by stating: "Progress over the last three years and the discussions at the current summit, however, suggest that it is time to define a rigorous, responsible translational pathway toward such trials". [ 64 ]  Inciting that the ethical and legal aspects should indeed be revisited G. Daley, representative of the summit's management and Dean of Harvard Medical School depicted Dr. He's experiment as "a wrong turn on the right path". [ 64 ]


The experiment was met with widespread criticism and was very controversial, globally as well as in China. [ 65 ] [ 66 ]  Several  bioethicists , researchers and medical professionals have released statements condemning the research, including  Nobel laureate   David Baltimore  who deemed the work "irresponsible" and one pioneer of the CRISPR/Cas9 technology, biochemist  Jennifer Doudna  at  University of California, Berkeley . [ 60 ] [ 67 ]  The director of the NIH,  Francis S. Collins  stated that the "medical necessity for inactivation of CCR5 in these infants is utterly unconvincing" and condemned He Jiankui and his research team for 'irresponsible work'. [ 61 ]  Other scientists, including  geneticist   George Church  of  Harvard University  suggested gene editing for disease resistance was "justifiable" but expressed reservations regarding the conduct of He's work. [ 68 ]


The Safe Genes program by DARPA has the goal to protect soldiers against gene editing war tactics. [ 69 ]  They receive information from ethical experts to better predict and understand future and current potential gene editing issues. [ 69 ] [ non-primary source needed ]


The  World Health Organization  has launched a global registry to track research on human genome editing, after a call to halt all work on genome editing. [ 70 ] [ 71 ] [ 72 ]


The  Chinese Academy of Medical Sciences  responded to the controversy in the journal  Lancet , condemning He for violating ethical guidelines documented by the government and emphasising that germline engineering should not be performed for reproductive purposes. [ 73 ]  The academy ensured they would "issue further operational, technical and ethical guidelines as soon as possible" to impose tighter regulation on human embryo editing.


Editing embryos, germ cells and the generation of designer babies is the subject of ethical debate, as a result of the implications in modifying genomic information in a heritable manner. This includes arguments over unbalanced gender selection and gamete selection. [ 74 ]


Despite regulations set by individual countries' governing bodies, the absence of a standardized regulatory framework leads to frequent discourse in discussion of germline engineering among scientists, ethicists and the general public.  Arthur Caplan , the head of the Division of Bioethics at  New York University  suggests that establishing an international group to set guidelines for the topic would greatly benefit global discussion and proposes instating "religious and ethics and legal leaders" to impose well-informed regulations. [ 75 ]


In many countries, editing embryos and germline modification for reproductive use is illegal. [ 76 ]   As of 2017, the U.S. restricts the use of germline modification and the procedure is under heavy regulation by the FDA and NIH. [ 76 ]   The American  National Academy of Sciences  and  National Academy of Medicine  indicated they would provide qualified support for human germline editing "for serious conditions under stringent oversight", should safety and efficiency issues be addressed. [ 77 ]   In 2019, World Health Organization called human germline genome editing as "irresponsible". [ 78 ]


Since genetic modification poses risk to any  organism , researchers and medical professionals must give the prospect of germline engineering careful consideration. The main ethical concern is that these types of treatments will produce a change that can be passed down to future generations and therefore any error, known or unknown, will also be passed down and will affect the offspring. [ 79 ]   Theologian  Ronald Green  of  Dartmouth College  has raised concern that this could result in a decrease in  genetic diversity  and the accidental introduction of new diseases in the future. [ 80 ]


When considering support for research into germline engineering, ethicists have often suggested that it can be considered unethical not to consider a technology that could improve the lives of children who would be born with  congenital disorders . Geneticist George Church claims that he does not expect germline engineering to increase societal disadvantage, and recommends lowering costs and improving education surrounding the topic to dispel these views. [ 8 ]  He emphasizes that allowing germline engineering in children who would otherwise be born with congenital defects could save around 5% of babies from living with potentially avoidable diseases. Jackie Leach Scully, professor of social and  bioethics  at  Newcastle University , acknowledges that the prospect of designer babies could leave those living with diseases and unable to afford the technology feeling marginalized and without medical support. [ 8 ]  However, Professor Leach Scully also suggests that germline editing provides the option for parents "to try and secure what they think is the best start in life" and does not believe it should be ruled out. Similarly,  Nick Bostrom , an  Oxford   philosopher  known for his work on the risks of  artificial intelligence , proposed that "super-enhanced" individuals could "change the world through their creativity and discoveries, and through innovations that everyone else would use". [ 81 ]


Many bioethicists emphasize that germline engineering is usually considered in the best interest of a child, therefore associated should be supported.  Dr James Hughes , a bioethicist at  Trinity College, Connecticut , suggests that the decision may not differ greatly from others made by parents which are well accepted – choosing with whom to have a child and using contraception to denote when a child is conceived. [ 82 ]   Julian Savulescu , a bioethicist and philosopher at Oxford University believes parents "should allow selection for non‐disease genes even if this maintains or increases social inequality", coining the term  procreative beneficence  to describe the idea that the children "expected to have the best life" should be selected. [ 83 ]  The  Nuffield Council on Bioethics  said in 2017 that there was "no reason to rule out" changing the DNA of a human embryo if performed in the child's interest, but stressed that this was only provided that it did not contribute to societal inequality. [ 8 ]  Furthermore, Nuffield Council in 2018 detailed applications, which would preserve equality and benefit humanity, such as elimination of hereditary disorders and adjusting to warmer climate. [ 84 ]  Philosopher and Director of Bioethics at non-profit Invincible Wellbeing  David Pearce [ 85 ]  argues that "the question [of designer babies] comes down to an analysis of risk-reward ratios - and our basic ethical values, themselves shaped by our evolutionary past." According to Pearce,"it's worth recalling that each act of old-fashioned sexual reproduction is itself an untested genetic experiment", often compromising a child's wellbeing and pro-social capacities even if the child grows in a healthy environment. [ 86 ]  Pearce thinks that as technology matures, more people may find it unacceptable to rely on "genetic roulette of natural selection". [ 87 ]


Conversely, several concerns have been raised regarding the possibility of generating designer babies, especially concerning the inefficiencies currently presented by the technologies. Green stated that although the technology was "unavoidably in our future", he foresaw "serious errors and health problems as unknown genetic side effects in 'edited' children" arise. [ 88 ]  Furthermore, Green warned against the possibility that "the well-to-do" could more easily access the technologies "..that make them even better off". This concern regarding germline editing exacerbating a societal and financial divide is shared amongst other researches, with the chair of the Nuffield Bioethics Council Professor Karen Yeung stressing that if funding of the procedures "were to exacerbate social injustice, in our view that would not be an ethical approach". [ 8 ]


Social and religious worries also arise over the possibility of editing human embryos. In a survey conducted by the  Pew Research Centre , it was found that only a third of the Americans surveyed who identified as strongly  Christian  approved of germline editing. [ 89 ]  Catholic leaders are in the middle ground. This stance is because, according to  Catholicism, a baby is a gift from God, and Catholics believe that people are created to be perfect in God's eyes. Thus, altering the genetic makeup of an infant is unnatural. In 1984, Pope John Paul II addressed that genetic manipulation in aiming to heal diseases is acceptable in the Church. He stated that it "will be considered in principle as desirable provided that it tends to the real promotion of the personal well-being of man, without harming his integrity or worsening his life conditions". [ 90 ]  However, it is unacceptable if designer babies are used to create a super/superior race including cloning humans. The Catholic Church rejects human cloning even if its purpose is to produce organs for therapeutic usage. The Vatican has stated that "The fundamental values connected with the techniques of artificial human procreation are two: the life of the human being called into existence and the special nature of the transmission of human life in marriage". [ 91 ]  According to them, it violates the dignity of the individual and is morally illicit.


A survey conducted by the Mayo Clinic in the Midwestern United States in 2017 saw that most of the participants agreed against the creation of designer babies with some noting its eugenic undertones. [ 92 ]  The participants also felt that gene editing may have unintended consequences that it may be manifested later in life for those that undergo gene editing. [ 92 ]  Some that took the survey worried that gene editing may lead to a decrease in the genetic diversity of the population in societies. [ 92 ]  The survey also noted how the participants were worried about the potential socioeconomic effects designer babies may exacerbate. [ 92 ]  The authors of the survey noted that the results of the survey showed that there is a greater need for interaction between the public and the scientific community concerning the possible implications and the recommended regulation of gene editing as it was unclear to them how much those that participated knew about gene editing and its effects prior to taking the survey. [ 92 ]


In Islam, the positive attitude towards genetic engineering is based on the general principle that Islam aims at facilitating human life. However, the negative view comes from the process used to create a designer baby. Oftentimes, it involves the destruction of some embryos. Muslims believe that "embryos already has a soul" at conception. [ 93 ]  Thus, the destruction of embryos is against the teaching of the Qur'an, Hadith, and Shari'ah law, that teaches our responsibility to protect human life. To clarify, the procedure would be viewed as "acting like God/Allah".  With the idea, that parents could choose the gender of their child, Islam believes that humans have no decision to choose the gender, and that "gender selection is only up to God". [ 94 ] [ contradictory ]


Since 2020, there have been discussions about American studies that use embryos without embryonic implantation with the CRISPR/Cas9 technique that had been modified with HDR (homology-directed repair), and the conclusions from the results were that gene editing technologies are currently not mature enough for real world use and that there is a need for more studies that generate safe results over a longer period of time. [ 95 ]


An article in the journal  Bioscience Reports  discussed how health in terms of genetics is not straightforward and thus there should be extensive deliberation for operations involving gene editing when the technology gets mature enough for real world use, where all of the potential effects are known on a case-by-case basis to prevent undesired effects on the subject or patient being operated on. [ 96 ]


Social aspects also raise concern, as highlighted by Josephine Quintavelle, director of  Comment on Reproductive Ethics  at  Queen Mary University of London , who states that selecting children's traits is "turning parenthood into an unhealthy model of self-gratification rather than a relationship". [ 97 ]


One major worry among scientists, including  Marcy Darnovsky  at the  Center for Genetics and Society  in  California , is that permitting germline engineering for correction of disease phenotypes is likely to lead to its use for cosmetic purposes and enhancement. [ 8 ]  Meanwhile,  Henry Greely , a bioethicist at  Stanford University  in California, states that "almost everything you can accomplish by gene editing, you can accomplish by embryo selection", suggesting the risks undertaken by germline engineering may not be necessary. [ 88 ]  Alongside this, Greely emphasizes that the beliefs that genetic engineering will lead to enhancement are unfounded, and that claims that we will enhance intelligence and personality are far off – "we just don't know enough and are unlikely to for a long time – or maybe for ever".


DNA-directed RNA interference  (ddRNAi) is a  gene-silencing  technique that utilizes  DNA constructs  to activate a cell's  endogenous   RNA interference  (RNAi) pathways. DNA constructs are designed to  express  self-complementary  double-stranded RNAs , typically  short-hairpin RNAs  (shRNA), that bring about the silencing of a target  gene  or genes once processed. [ 1 ]  Any  RNA , including endogenous  messenger RNA  (mRNAs) or  viral RNAs , can be silenced by designing constructs to express double-stranded RNA complementary to the desired  mRNA  target.


This mechanism has been recently demonstrated to work therapeutically to silence disease-causing genes across a range of disease models, including  viral diseases  such as  HIV , [ 2 ]   hepatitis B [ 3 ]  or  hepatitis C , [ 4 ]  and diseases associated with altered expression of endogenous genes such as drug-resistant  lung cancer , [ 5 ]   neuropathic pain , [ 6 ]   advanced cancer , [ 7 ]  and  retinitis pigmentosa . [ 8 ]


Unlike  small interfering RNAs  (siRNA) that turn over within a cell and silence genes transiently, DNA constructs are continually transcribed, replenishing the cellular supply of siRNAs. This allows for the long-term silencing of targeted genes, which has the potential for ongoing clinical benefit with reduced medical intervention. [ 2 ] [ 4 ]


Figure 1 illustrates the most common type of ddRNAi DNA construct designed to express a shRNA. This figure consists of a  promoter  sequence driving the expression of  sense  and  antisense  sequences separated by a loop sequence, followed by a transcriptional  terminator . The antisense sequence processed from the shRNA can bind to the target RNA and specify its degradation. shRNA constructs typically encode sense and antisense sequences of 20–30  nucleotides . Flexibility in construct design is possible; for example, the positions of sense and antisense sequences can be reversed, and other modifications and additions can alter intracellular shRNA processing. [ 9 ]  Moreover, a variety of promoter loop and terminator sequences can be used. [ 4 ]


A variant of this is the  multi-cassette  (Figure 2b). Designed to express two or more shRNAs, it can target multiple sequences for degradation simultaneously, which can be beneficial in circumstances such as when targeting viruses. Natural sequence variations can render a single shRNA-target site unrecognizable, preventing RNA degradation. Multi-cassette constructs that target multiple sites within the same viral RNA circumvent this issue. [ 4 ]


Delivery of ddRNAi DNA constructs is a major challenge for RNAi-based therapy. There are a number of clinically-approved  gene therapy  vectors developed for therapeutic use. Two broad strategies to facilitate the delivery of DNA constructs to the desired cells are available: these use either viral vectors or one of several classes of  transfection  reagents.


In vivo   delivery of ddRNAi constructs has been demonstrated using a range of vectors and reagents with different  routes of administration  (ROA). [ 3 ] [ 4 ] [ 6 ] [ 8 ]  ddRNAi constructs have also been successfully delivered into host cells  ex vivo , and then transplanted back into the host. [ 2 ] [ 7 ] [ 10 ]


For example, in a phase I clinical trial at the  City of Hope National Medical Center , California, four HIV-positive patients with  non-Hodgkin's lymphoma  were successfully treated with autologous  hematopoietic progenitor cells  pre-transduced ex vivo with ddRNAi constructs using  lentiviral vectors . This construct was designed to express three therapeutic RNAs, one of which was a shRNA, thereby combating HIV replication in three different ways: [ 2 ]


Ongoing expression of the shRNA has been confirmed in  T cells ,  monocytes , and  B cells  more than one year after transplantation. [ 2 ]


Nervana is an investigational V construct that  knocks down  the expression of  protein kinase C  gamma (PKCγ) known to be associated with  neuropathic pain  and  morphine tolerance . [ 6 ]


Two conserved PKCγ sequences across all key model species and humans have been identified, and both single and double DNA cassettes are designed.  In vitro , the expression of PKCγ was silenced by 80%. When similar ddRNAi constructs were delivered intrathecally using a lentiviral vector, pain relief in a neuropathic-rat model was demonstrated. [ 6 ]


The development of resistance to  chemotherapies  such as  paclitaxel  and  cisplatin  in  non-small-cell lung cancer  (NSCLC) is strongly associated with overexpression of  beta III tubulin . Investigations by the Children's Cancer Institute Australia ( University of New South Wales, Lowy Cancer Research Centre ) demonstrated that beta III-tubulin knockdown by ddRNAi delayed  tumor  growth and increased  chemosensitivity  in mouse models. [ 5 ]


Tributarna is a triple DNA cassette expressing three shRNA molecules each of which separately targets beta III tubulin and strongly inhibits its expression. Studies in an orthotopic-mouse model, where the construct is delivered by a modified  polyethylenimine  vector, jetPEI, that targets lung tissue are in progress. [ citation needed ]