The United States Government has made it clear that biosafety is to be taken very seriously. [ 31 ]   In 2014, incidents with anthrax and Ebola pathogens in CDC laboratories [ 32 ] [ 33 ]  prompted the CDC director  Tom Frieden  to issue a  moratorium  for research with these types of  select agents .  An investigation concluded that there was a lack of adherence to safety protocols and "inadequate safeguards" in place.  This indicated a lack of proper training or reinforcement of training and supervision on regular basis for lab personnel.


Following these incidents, the CDC established an External Laboratory Safety Workgroup (ELSW), [ 34 ]  and suggestions have been made to reform effectiveness of the Federal Select Agent Program. [ 35 ]  The White House issued a report on national biosafety priorities in 2015, outlining next steps for a national biosafety and security program, and addressed biological safety needs for health research, national defense, and public safety. [ 36 ]


In 2016, the Association of Public Health Laboratories (APHL) had a presentation at their annual meeting focused on improving biosafety culture. [ 37 ]  This same year, The UPMC Center for Health Security issued a case study report including reviews of ten different nations' current biosafety regulations, including the United States.  Their goal was to "provide a foundation for identifying national-level biosafety norms and enable initial assessment of biosafety priorities necessary for developing effective national biosafety regulation and oversight." [ 38 ]


Blasticidin S  is an  antibiotic  that is used in biology research for  selecting  cells in  cell culture . Cells of interest can express the blasticidin resistance genes BSD or bsr, and can then survive treatment with the antibiotic. Blasticidin S is a  nucleoside analogue  antibiotic, resembling the nucleoside  cytidine . Blasticidin works against human cells, fungi, and bacteria, all by disrupting  protein translation . It was originally described by Japanese researchers in the 1950s seeking antibiotics for  rice blast fungus .


A nucleoside analog, blasticidin S resembles the nucleoside  cytidine . The chemical structure consists of a  cytosine  molecule, linked to a  glucuronic acid -derived ring, linked in turn to the  peptide  N-methyl β-arginine. [ 1 ]


Blasticidin S is widely used in  cell culture  for  selecting  and maintaining genetically manipulated cells. Cells of interest express the blasticidin S resistance genes BSD or bsr, and can then survive blasticidin S being added to the  culture media . [ 2 ]  Blasticidin S is typically used at 2–300  micrograms  per milliliter of media, depending on the type of cell being grown. [ 2 ]


Blasticidin prevents the growth of both  eukaryotic  and  prokaryotic  cells.  It works by inhibiting termination step of translation and peptide bond formation (to lesser extent) by the  ribosome .  This means that cells can no longer produce new proteins through  translation  of  mRNA . It is competitive with  puromycin  suggesting a highly similar binding site. [ 3 ]


The first step in blasticidin S biosynthesis is the combination of  UDP-glucuronic acid  with cytosine to form cytosylglucuronic acid (CGA). Given the product name, the enzyme that performs this combination is called  CGA synthase . [ 1 ]


Cosmid cloning experiments from the Blasticidin S producer  Streptomyces griseochromogenes , followed by evaluation of the putative biosynthetic gene cluster via heterologous reconstitution of Blasticidin S production in  Streptomyces lividans , indicated that a 20 Kbp gene cluster with 19 genes, plus possibly a peptidase outside the gene cluster that acts on the final leucylblasticidin
S (LBS) intermediate, was sufficient for reconstitution of Blasticidin S biosynthesis. [ 3 ]


Resistance  to blasticidin S can be conferred by either of two  deaminases : BSD, originally isolated from  Aspergillus terreus  or bsr, isolated from  Bacillus cereus . Both deaminases work by modifying blasticidin S directly, replacing the amine on the cytosine ring with a hydroxyl group, resulting in the inactive deaminohydroxy-blasticin S. [ 2 ] [ 4 ]


bsr and BSD are the most commonly used resistance genes.  The proteins produced from these genes enable the cells carrying them to produce proteins in the presence of blasticidin.


In the 1950s, a drug screening program was designed in  Japan  to discover a new antibiotic that prevents  blast disease  by the  fungus   Magnaporthe grisea . [ 5 ]


Emmanuelle Marie Charpentier  ( French pronunciation:   [emanɥɛl   maʁi   ʃaʁpɑ̃tje] ; born 11 December 1968 [ 2 ] ) is a French professor and researcher in  microbiology ,  genetics , and  biochemistry . [ 1 ]  As of 2015, she has been a director at the  Max Planck Institute for Infection Biology  in  Berlin . In 2018, she founded an independent  research institute , the Max Planck Unit for the Science of Pathogens. [ 3 ]  In 2020, Charpentier and American biochemist  Jennifer Doudna  of the  University of California, Berkeley , were awarded the  Nobel Prize in Chemistry  "for the development of a method for  genome editing " (through  CRISPR ). This was the first science Nobel Prize ever won by two women only. [ 4 ] [ 5 ] [ 6 ]


Born in 1968 in  Juvisy-sur-Orge  in France, Charpentier studied biochemistry, microbiology, and genetics at the  Pierre and Marie Curie University  (which became the Faculty of Science of  Sorbonne University ) in Paris. [ 7 ]  She was a graduate student at the  Institut Pasteur  from 1992 to 1995 and was awarded a research doctorate. Charpentier's PhD work investigated molecular mechanisms involved in  antibiotic resistance . [ 8 ]  Her paternal grandfather, surnamed Sinanian, was an  Armenian  who escaped to France during the  Armenian Genocide  and met his wife in  Marseille . [ 9 ]


Charpentier worked as a university teaching assistant at Pierre and Marie Curie University from 1993 to 1995 and as a postdoctoral fellow at the  Institut Pasteur  from 1995 to 1996. She moved to the US and worked as a postdoctoral fellow at  Rockefeller University  in New York from 1996 to 1997. During this time, Charpentier worked in the lab of microbiologist  Elaine Tuomanen . [ 10 ]  Tuomanen's lab investigated how the pathogen  Streptococcus pneumoniae  utilizes mobile genetic elements to alter its genome. Charpentier also helped to demonstrate how  S. pneumoniae  develops  vancomycin  resistance. [ 11 ]


Charpentier was an assistant research scientist at the  New York University Medical Center  from 1997 to 1999. She worked in the lab of Pamela Cowin, a skin-cell biologist interested in mammalian gene manipulation. Charpentier published a paper exploring the regulation of hair growth in mice. [ 12 ]  She held the position of Research Associate at the  St. Jude Children's Research Hospital  and at the  Skirball Institute of Biomolecular Medicine [ 13 ]  in New York from 1999 to 2002. [ 7 ]


After five years in the United States, Charpentier returned to Europe and became the lab head and a guest professor at the Institute of Microbiology and Genetics,  University of Vienna , from 2002 to 2004. In 2004, Charpentier published her discovery of an RNA molecule involved in the regulation of virulence-factor synthesis in  Streptococcus pyogenes . [ 14 ]  From 2004 to 2006 she was lab head and an assistant professor at the Department of Microbiology and Immunobiology. In 2006 she became a  privatdozentin  (Microbiology) and received her  habilitation  at the Centre of Molecular Biology. From 2006 to 2009 she worked as lab head and associate professor at the  Max F. Perutz Laboratories . [ 7 ]


Charpentier moved to Sweden and became lab head and associate professor at the Laboratory for  Molecular Infection Medicine Sweden  (MIMS), at  Umeå University . She held the position of group leader from 2008 to 2013 and was visiting professor from 2014 to 2017. [ 15 ]  She moved to Germany to act as department head and W3 Professor at the  Helmholtz Centre for Infection Research [ 16 ]  in Braunschweig and the  Hannover Medical School  from 2013 until 2015. In 2014 she became an  Alexander von Humboldt Professor . [ 7 ]


In 2015 Charpentier accepted an offer from the German  Max Planck Society  to become a scientific member of the society and a director at the  Max Planck Institute for Infection Biology  in  Berlin . Since 2016, she has been an Honorary Professor at  Humboldt University  in Berlin; since 2018, she is the Founding and acting director of the  Max Planck Unit for the Science of Pathogens . [ 7 ] [ 17 ] [ 18 ]  Charpentier retained her position as visiting professor at Umeå University until the end of 2017 when a new donation from the Kempe Foundations and the  Knut and Alice Wallenberg Foundation  allowed her to offer more young researchers positions within research groups of the MIMS Laboratory. [ 19 ]


Charpentier is best known for her Nobel-winning work of deciphering the molecular mechanisms of a bacterial immune system, called  CRISPR / Cas9 , and repurposing it into a tool for  genome editing . In particular, she uncovered a novel mechanism for the maturation of a non-coding RNA which is pivotal in the function of CRISPR/Cas9. Specifically, Charpentier demonstrated that a small RNA called  tracrRNA  is essential for the maturation of crRNA. [ 20 ]


In 2011, Charpentier met  Jennifer Doudna  at a research conference in San Juan, Puerto Rico, and they began a collaboration. [ 10 ]  Working with Doudna's laboratory, Charpentier's laboratory showed that Cas9 could be used to make cuts in any  DNA  sequence desired. [ 21 ] [ 22 ]  The method they developed involved the combination of Cas9 with easily created synthetic "guide RNA" molecules. Synthetic guide RNA is a chimera of crRNA and tracrRNA; therefore, this discovery demonstrated that the CRISPR-Cas9 technology could be used to edit the genome with relative ease. [ 22 ]  Researchers worldwide have employed this method successfully to edit the DNA sequences of plants, animals, and laboratory  cell lines . Since its discovery, CRISPR has revolutionized genetics by allowing scientists to edit genes to probe their role in health and disease and to develop genetic therapies with the hope that it will prove safer and more effective than the first generation of gene therapies. [ 6 ]


In 2013, Charpentier co-founded  CRISPR Therapeutics  and ERS Genomics along with Shaun Foy and Rodger Novak. [ 23 ]


In 2015,  Time  magazine designated Charpentier one of the  Time  100  most influential people in the world (together with Jennifer Doudna). [ 24 ] [ 25 ]


Charpentier's awards are:


Nobel Prize in Chemistry , [ 26 ]  the  Breakthrough Prize  in  Life Sciences , the  Louis-Jeantet Prize for Medicine , the  Gruber Foundation  International Prize in Genetics, the  Leibniz Prize , the  Tang Prize , the  Japan Prize , and the  Kavli Prize  in Nanoscience. She has won the  BBVA Foundation Frontiers of Knowledge Award  jointly with  Jennifer Doudna  and  Francisco Mojica . [ 27 ]


In 2019, Charpentier was a featured character in the play  STEM FEMMES  by Philadelphia theater company Applied Mechanics. [ 89 ]


In 2021,  Walter Isaacson  detailed the story of Jennifer Doudna and her collaboration with Charpentier leading to the discovery of CRISPR/CAS-9, in the biography  The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race . [ 90 ]


Chemically induced dimerization  ( CID ) is a biological mechanism in which two proteins bind only in the presence of a certain small molecule, enzyme or other dimerizing agent. [ 1 ]  Genetically engineered CID systems are used in biological research to control protein localization, to manipulate  signalling pathways  and to induce protein activation. [ 2 ]


The first small molecule CID system was developed in 1993 and used  FK1012 , a derivative of the drug  tacrolimus  (FK506), to induce homo-dimerization of  FKBP . [ 2 ]  This system was used  in vivo  to induce binding between cell surface receptors which could not bind in the normal way because they lacked the transmembrane and extracellular domain. Addition of FK1012 to the cells caused signal transduction.


CID has been used for a number of applications in biomedical research. In most applications each dimerizing protein is expressed as part of a fusion construct with other proteins of interest. Adding the chemical dimerizing agent brings both  constructs  into proximity with each other and induces interactions between the proteins of interest. CID has been used to regulate and monitor gene transcription,  signal transduction  and post translational modifications in proteins.


Recently, CID has also been used to create a basic component of  biocomputers , logic gates, from genetically manipulated cells. [ 8 ]  In this application, two independent CID systems, one based on plant proteins and one based on bacterial proteins are expressed in the same cell. Each set of proteins can be induced to dimerize by the addition of a separate chemical. By creating  fusion proteins  with the dimerizing proteins, membrane bound proteins and proteins that activate cell ruffling an  AND gate  and  OR gate  can be created that take chemical dimerizing agents as inputs and returns a ruffled or unruffled state as output.


Cisgenesis  is a product designation for a category of  genetically engineered  plants. A variety of  classification schemes  have been proposed [ 1 ]  that order  genetically modified organisms  based on the nature of introduced genotypical changes, rather than the process of genetic engineering.


Cisgenesis (etymology: cis = same side; and genesis = origin) is one term for organisms that have been engineered using a process in which genes are artificially transferred between organisms that could otherwise be conventionally bred. [ 2 ] [ 3 ]   Genes  are only transferred between closely related organisms. [ 4 ]  Nucleic acid sequences must be isolated and introduced using the same technologies that are used to produce transgenic organisms, making cisgenesis similar in nature to transgenesis. The term was first introduced in 2000 by Henk J. Schouten and Henk Jochemsen, [ 5 ]  and in 2004 a PhD thesis by Jan Schaart of  Wageningen University  in 2004, discussing making  strawberries  less susceptible to  Botrytis cinerea .


In  Europe , currently, this process is governed by the same laws as transgenesis. While researchers at Wageningen University in the  Netherlands  feel that this should be changed and regulated in the same way as conventionally  bred plants , other scientists, writing in  Nature Biotechnology , have disagreed. [ 3 ]  In 2012 the  European Food Safety Authority  (EFSA) issued a report with their risk assessment of cisgenic and intragenic plants. They compared the hazards associated with plants produced by cisgenesis and intragenesis with those obtained either by conventional plant breeding techniques or transgenesis. The EFSA concluded that "similar hazards can be associated with cisgenic and conventionally bred plants, while novel hazards can be associated with intragenic and transgenic plants." [ 6 ]


Cisgenesis has been applied to transfer of natural resistance genes to the devastating disease  Phytophthora infestans  in potato [ 7 ]  and  scab  ( Venturia inaequalis ) in apple. [ 8 ] [ 9 ]


Cisgenesis and transgenesis use artificial  gene transfer , which results in less extensive change to an organism's genome than  mutagenesis , which was widely used before genetic engineering was developed. [ 10 ]


Some people believe that cisgenesis should not face as much regulatory oversight as  genetic modification  created through transgenesis as it is possible, if not practical, to transfer alleles among closely related species even by traditional crossing. The primary biological advantage of cisgenesis is that it does not disrupt favorable heterozygous states, particularly in asexually propagated crops such as potato, which do not breed true to seed. One application of cisgenesis is to create  blight  resistant potato plants by transferring known resistance loci wild genotypes into modern, high yielding varieties. [ 11 ]


The Dutch government has proposed to exclude cisgenic plants from the European GMO Regulation, in view of the safety of cisgenic plants compared to classically bred plants, and their contribution to durable food production. [ 12 ]


A related classification scheme proposed by Kaare Nielsen is: [ 1 ]


Conditional gene knockout  is a technique used to eliminate a specific gene in a certain tissue, such as the liver. [ 1 ] [ 2 ]  This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. Using the conditional gene knockout technique eliminates many of the side effects from traditional  gene knockout . In traditional gene knockout,  embryonic death  from a gene  mutation  can occur, and this prevents scientists from studying the gene in adults. Some tissues cannot be studied properly in isolation, so the gene must be inactive in a certain tissue while remaining active in others. With this technology, scientists are able to knockout genes at a specific stage in development and study how the knockout of a gene in one tissue affects the same gene in other tissues. [ 3 ] [ 4 ]


The most commonly used technique is the Cre-lox recombination system. The Cre recombinase enzyme specifically recognizes two lox (loci of recombination) sites within DNA and causes  recombination  between them. During recombination two strands of DNA exchange information. This recombination will cause a deletion or inversion of the genes between the two lox sites, depending on their orientation. An entire gene can be removed to inactivate it. [ 1 ] [ 3 ]  This whole system is inducible so a chemical can be added to knock genes out at a specific time. Two of the most commonly used chemicals are tetracycline, which activates transcription of the Cre recombinase gene and tamoxifen, which activates transport of the Cre recombinase protein to the nucleus. [ 4 ]  Only a few cell types express Cre recombinase and no mammalian cells express it so there is no risk of accidental activation of lox sites when using conditional gene knockout in mammals. Figuring out how to express Cre-recombinase in an organism tends to be the most difficult part of this technique. [ 3 ]


The conditional gene knockout method is often used to model human diseases in other mammals. [ 2 ]  It has increased scientists’ ability to study diseases, such as cancer, that develop in specific cell types or developmental stages. [ 4 ]  It is known that mutations in the BRCA1 gene are linked to breast cancer. Scientists used conditional gene knockout to delete the BRCA1  allele  in mammary gland tissue in mice and found that it plays an important role in tumour suppression. [ 3 ]


A specific gene in  mouse brain  thought to be involved in the onset of  Alzheimer's disease  which codes for the enzyme  cyclin-dependent kinase 5  (Cdk5) was knocked out. Such mice were found to be 'smarter' than normal mice and were able to handle complex tasks more intelligently compared to 'normal' mice bred in the laboratory. [ 5 ]


Conditional gene knockouts in mice are often used to study human diseases because many genes produce similar phenotypes in both species. For the past 100 years laboratory mouse genetics have been used for this because mice are mammals that are physiologically similar enough to humans to generate qualitative testing. These two have such similar genes that out of 4000 studied genes, only 10 were found in one species but not the other.  All mammals shared the same common ancestor approximately 80 million years ago; technically speaking, all genomes of mammals are comparatively similar. However, in comparison between mice and humans, their protein-coding regions of the genomes are 85% identical and have similarities between 99% of their homologs. These similarities result in similar phenotypes to be expressed between the two species.[8][12] Their genes are very alike to those of humans with 99% having homologs being similar. Along with producing similar phenotypes as well making them very promising candidates for conditional gene knockouts.[8] The goal of KOMP is to create knockout mutations in the embryonic stem cells for each of the 20,000 protein coding genes in mice. [ 2 ]  The genes are knocked out because this is the best way to study their function and learn more about their role in human diseases. There are two main strategies to conditional gene knockout and those are gene targeting or homologous recombination and gene trapping. Both methods usually have a modified viral vector or a linear fragment as the mode of transportation of the artificial DNA into the target ES cell. The cells then grow in a petri dish for several days and are inserted into the early-stage embryos. Lastly, the embryos are placed into the adult female's uterus where it can grow into its offspring.[9] Some alleles in this project cannot be knocked out using traditional methods and require the specificity of the conditional gene knockout technique. Other combinatorial methods are needed to knockout the last remaining alleles. Conditional gene knockout is a time-consuming procedure and there are additional projects focusing on knocking out the remaining mouse genes. [ 6 ]  The KOMP project contributor,  Oliver Smithies , arguably provided the biggest scientific impact on this gene targeting. Oliver received the Nobel prize for medicine due to a technique allowing the ability to identify functions in genes and how to use the 'knockout' method to delete certain genes. Unfortunately, the pioneer in gene targeting died at the age of 91 on January 10, 2017.[11] The KOMP projected was started in 2006 and is still ongoing today. [ 7 ]  The KOMP Repository provides incentives to those partaking in the projects to return feedback to them and those who meet specific criteria can be refunded 50% of the cost of their research cells.[10]


8. Austin, C. P., Battey, J. F., Bradley, A., Bucan, M., Capecchi, M., Collins, F. S., Dove, W. F., Duyk, G., Dymecki, S., Eppig, J. T., Grieder, F. B., Heintz, N.,         Hicks, G., Insel, T. R., Joyner, A., Koller, B. H., Lloyd, K. C., Magnuson, T., Moore, M. W., Nagy, A., ... Zambrowicz, B. (2004). The knockout mouse project. Nature genetics, 36(9), 921–924. https://doi.org/10.1038/ng0904-921


9. Knockout Mice Fact Sheet. (n.d.). Retrieved from https://www.genome.gov/about-genomics/fact-sheets/Knockout-Mice-Fact-Sheet


10. Lloyd K. C. (2011). A knockout mouse resource for the biomedical research community. Annals of the New York Academy of Sciences, 1245, 24–26. https://doi.org/10.1111/j.1749-6632.2011.06311.x


11. Nobel Prize winner Dr. Oliver Smithies to deliver Earl H. Morris Endowed Lecture on July 10. (n.d.). Retrieved from https://medicine.wright.edu/about/article/2009/smithieslecture


12. NIH. (n.d.). Why Mouse Matters. Retrieved from https://www.genome.gov/10001345/importance-of-mouse-genome


A  cosmid  is a type of hybrid  plasmid  that contains a  Lambda phage   cos  sequence. [ 1 ]  Often used as  cloning vectors  in  genetic engineering , cosmids can be used to build  genomic libraries . They were first described by Collins and Hohn in 1978. [ 2 ]
Cosmids can contain 37 to 52 (normally 45)  kb  of DNA, limits based on the normal bacteriophage packaging size. They can  replicate  as plasmids if they have a suitable  origin of replication  (ori): for example  SV40  ori in mammalian cells,  ColE1  ori for double-stranded DNA replication, or  f1  ori for single-stranded DNA replication in  prokaryotes . They frequently also contain a gene for selection such as  antibiotic resistance , so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow. [ 3 ]


Unlike plasmids, they can also be packaged in vitro into  phage   capsids , a step which requires  cohesive ends , also known as  cos  sites also used in cloning with a lambda phage as a vector, however nearly all the lambda genes have been deleted with the exception of the  cos  sequence. The hybrid cosmid DNA in the capsids can then be transferred into bacterial cells by  transduction . Since there is a requirement for in vitro packaging whereby at least 38 kb of DNA is required between the cos sites, the vector without insert DNA will not be packaged (plasmids instability is increased if the novel inserted DNA contains many direct repeats or palindromic (inverted repeats) DNA. This instability can largely be counteracted by using a host bacterium with specific mutations affecting DNA recombination (N.B. Absence of inverted repeats was noted in the first Hohn & Collins publication cited above; see also [ 4 ] ).


Cos  sequences are ~200 base pairs long and essential for packaging. They contain a  cosN  site where DNA is nicked at each strand, 12 bp apart, by  terminase . This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The DNA must be linear to fit into a phage head.) The  cosB  site holds the terminase while it is nicking and separating the strands. The  cosQ  site of next cosmid (as  rolling circle replication  often results in linear  concatemers ) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular  DNases .


Cosmids are predominantly plasmids with a bacterial  oriV , an antibiotic selection marker and a cloning site, but they carry one, or more recently two, cos sites derived from bacteriophage lambda. Depending on the particular aim of the experiment, broad host range cosmids, shuttle cosmids or 'mammalian' cosmids (linked to SV40 oriV and mammalian selection markers) are available. The loading capacity of cosmids varies depending on the size of the vector itself but usually lies around 40–45 kb. The cloning procedure involves the generation of two vector arms which are then joined to the foreign DNA. Selection against wild type cosmid DNA is simply done via size exclusion. Cosmids, therefore, always form colonies and not plaques. Also the clone density is much lower with around 10 5  – 10 6   CFU  per μg of ligated DNA.


After the construction of recombinant lambda or cosmid libraries the total DNA is transferred into an appropriate  E. coli  host via a technique called in vitro packaging. The necessary packaging extracts are derived from  E. coli  cI857 lysogens (red- gam- Sam and Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize and package the recombinant molecules  in vitro , generating either mature phage particles (lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). These differences are reflected in the different infection frequencies seen in favour of lambda-replacement vectors. This compensates for their slightly lower loading capacity. Phage libraries are also stored and screened more easily than cosmid libraries.


Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments. This is usually done by partial restriction followed by either size fractionation or dephosphorylation (using calf-intestine phosphatase) to avoid chromosome scrambling, i.e. the ligation of physically unlinked fragments.


Commonly used vectors include "SuperCos 1"


CRISPR  ( / ˈ k r ɪ s p ər / ) (an  acronym  for  c lustered  r egularly  i nterspaced  s hort  p alindromic  r epeats) is a family of  DNA  sequences found in the  genomes  of  prokaryotic  organisms such as  bacteria  and  archaea . [ 2 ]  Each sequence within an individual prokaryotic cell is derived from a DNA fragment of a  bacteriophage  that had previously infected the prokaryote or one of its ancestors. [ 3 ] [ 4 ]  These sequences are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti- phage ) defense system of prokaryotes and provide a form of heritable, [ 3 ]   acquired immunity . [ 2 ] [ 5 ] [ 6 ] [ 7 ]  CRISPR is found in approximately 50% of sequenced  bacterial genomes  and nearly 90% of sequenced archaea. [ 3 ]


Cas9  (or "CRISPR-associated protein 9") is an  enzyme  that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as  CRISPR-Cas9  that can be used to edit genes within living organisms. [ 9 ] [ 10 ]  This editing process has a wide variety of applications including basic biological research, development of  biotechnological  products, and treatment of diseases. [ 11 ] [ 12 ]  The development of the CRISPR-Cas9 genome editing technique was recognized by the  Nobel Prize in Chemistry  in 2020 awarded to  Emmanuelle Charpentier  and  Jennifer Doudna . [ 13 ] [ 14 ]


The discovery of clustered DNA repeats took place independently in three parts of the world. The first description of what would later be called CRISPR is from  Osaka University  researcher  Yoshizumi Ishino  and his colleagues in 1987. They accidentally cloned part of a CRISPR sequence together with the " iap" gene   (isozyme conversion of alkaline phosphatase)  from their target genome, that of  Escherichia coli . [ 15 ] [ 16 ]  The organization of the repeats was unusual. Repeated sequences are typically arranged consecutively, without interspersing different sequences. [ 12 ] [ 16 ]  They did not know the function of the interrupted clustered repeats.


In 1993, researchers of  Mycobacterium tuberculosis  in the Netherlands published two articles about a cluster of interrupted  direct repeats  (DR) in that bacterium. They recognized the diversity of the sequences that intervened in the direct repeats among different strains of  M. tuberculosis [ 17 ]  and used this property to design a typing method called  spoligotyping , still in use today. [ 18 ] [ 19 ]


Francisco Mojica  at the  University of Alicante  in Spain studied the function of repeats in the archaeal species  Haloferax  and  Haloarcula . Mojica's supervisor surmised that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division, because plasmids and chromosomes with identical repeat arrays could not coexist in  Haloferax volcanii . Transcription of the interrupted repeats was also noted for the first time; this was the first full characterization of CRISPR. [ 19 ] [ 20 ]  By 2000, Mojica and his students, after an automated search of published genomes, identified interrupted repeats in 20 species of microbes as belonging to the same family. [ 21 ]  Because those sequences were interspaced, Mojica initially called these sequences "short regularly spaced repeats" (SRSR). [ 22 ]  In 2001, Mojica and  Ruud Jansen , who were searching for an additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to unify the numerous acronyms used to describe these sequences. [ 20 ] [ 23 ]  In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome of  Archaeoglobus fulgidus  were transcribed into long RNA molecules subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. [ 24 ] [ 25 ]


In 2005,  yogurt  researcher  Rodolphe Barrangou  discovered that  Streptococcus thermophilus , after iterative phage infection challenges, develops increased phage resistance due to the incorporation of additional CRISPR spacer sequences. [ 26 ]  Barrangou's employer, the Danish food company Danisco, then developed phage-resistant  S. thermophilus  strains for yogurt production. Danisco was later bought by  DuPont , which owns about 50 percent of the global dairy culture market, and the technology spread widely. [ 27 ]


A major advance in understanding CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by four homologous genes that make up CRISPR-associated systems,  cas  1–4. The Cas proteins showed  helicase  and  nuclease   motifs , suggesting a role in the dynamic structure of the CRISPR loci. [ 28 ]  In this publication, the acronym CRISPR was used as the universal name of this pattern, but its function remained enigmatic.


In 2005, three independent research groups showed that some CRISPR spacers are derived from  phage  DNA and  extrachromosomal DNA  such as  plasmids . [ 32 ] [ 33 ] [ 34 ]  In effect, the spacers are fragments of DNA gathered from viruses that previously attacked the cell. The source of the spacers was a sign that the CRISPR- cas  system could have a role in adaptive immunity in  bacteria . [ 29 ] [ 35 ]  All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals. [ 36 ]


The first publication [ 33 ]  proposing a role of CRISPR-Cas in microbial immunity, by Mojica and collaborators at the  University of Alicante , predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the  RNA interference  system used by eukaryotic cells. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins. [ 37 ]


Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was an adaptive immune system was published. [ 6 ] [ 12 ]  A CRISPR region in  Streptococcus thermophilus  acquired spacers from the DNA of an infecting  bacteriophage . The researchers manipulated the resistance of  S. thermophilus  to different types of phages by adding and deleting spacers whose sequence matched those found in the tested phages. [ 38 ] [ 39 ]  In 2008, Brouns and Van der Oost identified a complex of Cas proteins called Cascade, that in  E. coli  cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules called  CRISPR RNA  (crRNA), which remained bound to the protein complex. [ 40 ]  Moreover, it was found that Cascade, crRNA and a helicase/nuclease ( Cas3 ) were required to provide a bacterial host with immunity against infection by a  DNA virus . By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting  dsDNA . That year Marraffini and Sontheimer confirmed that a CRISPR sequence of  S. epidermidis  targeted DNA and not RNA to prevent  conjugation . This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in  Pyrococcus furiosus . [ 12 ] [ 39 ]  A 2010 study showed that CRISPR-Cas cuts strands of both phage and plasmid DNA in  S. thermophilus . [ 41 ]


A simpler CRISPR system from  Streptococcus pyogenes  relies on the protein  Cas9 . The Cas9  endonuclease  is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA). [ 42 ] [ 43 ]  In 2012,  Jennifer Doudna  and  Emmanuelle Charpentier  re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a " single-guide RNA " that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. [ 44 ]  This contribution was so significant that it was recognized by the  Nobel Prize in Chemistry  in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for separation. [ 44 ]  Another collaboration comprising  Virginijus Šikšnys , Gasiūnas, Barrangou, and Horvath showed that Cas9 from the  S. thermophilus  CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system. [ 19 ]


Groups led by  Feng Zhang  and  George Church  simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time. [ 12 ] [ 45 ] [ 46 ]  It has since been used in a wide range of organisms, including baker's yeast ( Saccharomyces cerevisiae ), [ 47 ] [ 48 ] [ 49 ]  the  opportunistic pathogen   Candida albicans , [ 50 ] [ 51 ]  zebrafish ( Danio rerio ), [ 52 ]  fruit flies ( Drosophila melanogaster ), [ 53 ] [ 54 ]  ants ( Harpegnathos saltator [ 55 ]  and  Ooceraea biroi [ 56 ] ), mosquitoes ( Aedes aegypti [ 57 ] ), nematodes ( Caenorhabditis elegans ), [ 58 ]  plants, [ 59 ]  mice ( Mus musculus domesticus ) , [ 60 ] [ 61 ]  monkeys [ 62 ]  and human embryos. [ 63 ]


CRISPR has been modified to make programmable  transcription factors  that allows activation or silencing of targeted genes. [ 64 ]


The CRISPR-Cas9 system has been shown to make effective gene edits in Human  tripronuclear zygotes , as first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin ( HBB ) in 28 out of 54 embryos. Four out of the 28 embryos were successfully recombined using a donor template. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells. [ 65 ]


In 2015, the nuclease  Cas12a  (formerly called  Cpf1 [ 66 ] ) was characterized in the CRISPR-Cpf1 system of the bacterium  Francisella novicida . [ 67 ] [ 68 ]  Its original name, from a  TIGRFAMs   protein family  definition built in 2012, reflects the prevalence of its CRISPR-Cas subtype in the  Prevotella  and  Francisella  lineages. Cas12a showed several key differences from Cas9 including: causing a 'staggered' cut in double stranded DNA as opposed to the 'blunt' cut produced by Cas9, relying on a 'T rich'  PAM  (providing alternative targeting sites to Cas9), and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast, Cas9 requires both crRNA and a  trans-activating crRNA  (tracrRNA).


These differences may give Cas12a some advantages over Cas9. For example, Cas12a's small crRNAs are ideal for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9's sgRNAs. The sticky 5′ overhangs left by Cas12a can also be used for DNA assembly that is much more target-specific than traditional restriction enzyme cloning. [ 69 ]  Finally, Cas12a cleaves DNA 18–23 base pairs downstream from the PAM site. This means there is no disruption to the recognition sequence after repair, and so Cas12a enables multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 base pairs upstream of the PAM site, the NHEJ pathway results in  indel  mutations that destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage should cause an increased opportunity for the desired genomic editing to occur. [ 70 ]  A distinctive feature of Cas12a, as compared to Cas9, is that after cutting its target, Cas12a remains bound to the target and then cleaves other ssDNA molecules non-discriminately. [ 71 ]  This property is called "collateral cleavage" or "trans-cleavage" activity and has been exploited for the development of various diagnostic technologies. [ 72 ] [ 73 ]


In 2016, the nuclease  Cas13a  (formerly known as  C2c2 ) from the bacterium  Leptotrichia shahii  was characterized. Cas13 is an RNA-guided RNA endonuclease, which means that it does not cleave DNA, but only single-stranded RNA. Cas13 is guided by its crRNA to a ssRNA target and binds and cleaves the target. Similar to Cas12a, the Cas13 remains bound to the target and then cleaves other ssRNA molecules non-discriminately. [ 74 ]  This collateral cleavage property has been exploited for the development of various diagnostic technologies. [ 75 ] [ 76 ] [ 77 ]


In 2021, Dr. Hui Yang characterized novel miniature Cas13 protein (mCas13) variants, Cas13X and Cas13Y. Using a small portion of N gene sequence from SARS-CoV-2 as a target in characterization of mCas13, revealed the sensitivity and specificity of mCas13 coupled with RT-LAMP for detection of SARS-CoV-2 in both synthetic and clinical samples over other available standard tests like RT-qPCR (1 copy/μL). [ 78 ]


The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers. [ 79 ]  CRISPR repeats typically range in size from 28 to 37  base pairs  (bps), though there can be as few as 23 bp and as many as 55 bp. [ 80 ]  Some show  dyad symmetry , implying the formation of a  secondary structure  such as a  stem-loop  ('hairpin') in the RNA, while others are designed to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). [ 80 ]  New spacers can appear rapidly as part of the immune response to phage infection. [ 81 ]  There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array. [ 80 ]


Small clusters of  cas  genes are often located next to CRISPR repeat-spacer arrays. Collectively the 93  cas  genes are grouped into 35 families based on sequence similarity of the encoded proteins. 11 of the 35 families form the  cas  core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the  cas  core. [ 82 ]


CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI. [ 83 ]  The 6 system types are divided into 33 subtypes. [ 84 ]  Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of  cas  genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The  phylogeny  of Cas1 proteins generally agrees with the classification system, [ 85 ]  but exceptions exist due to module shuffling. [ 82 ]  Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components. [ 86 ] [ 87 ]  The sporadic distribution of the CRISPR-Cas subtypes suggests that the CRISPR-Cas system is subject to  horizontal gene transfer  during microbial  evolution .


CRISPR-Cas immunity is a natural process of bacteria and archaea. [ 104 ]  CRISPR-Cas prevents bacteriophage infection,  conjugation  and  natural transformation  by degrading foreign nucleic acids that enter the cell. [ 39 ]


When a  microbe  is invaded by a  bacteriophage , the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer.  Cas1  and  Cas2  are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of Cas1 or Cas2 stopped spacer acquisition, without affecting CRISPR immune response. [ 105 ] [ 106 ] [ 107 ] [ 108 ] [ 109 ]


Multiple Cas1 proteins have been characterised and their structures resolved. [ 110 ] [ 111 ] [ 112 ]  Cas1 proteins have diverse  amino acid  sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/ integrases  that bind to DNA in a sequence-independent manner. [ 86 ]  Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA- [ 113 ]  or (double strand) dsDNA- [ 114 ] [ 115 ]  specific  endoribonuclease  activity.


In the I-E system of  E. coli  Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. [ 116 ]  In this complex Cas2 performs a non-enzymatic scaffolding role, [ 116 ]  binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays. [ 117 ] [ 118 ] [ 119 ]  New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections. [ 120 ]  In  E. coli  a  histone like protein  called integration host factor ( IHF ), which binds to the leader sequence, is responsible for the accuracy of this integration. [ 121 ]  IHF also enhances integration efficiency in the type I-F system of  Pectobacterium atrosepticum . [ 122 ]  but in other systems, different host factors may be required [ 123 ]


Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3–5 bp) DNA sequences termed  protospacer adjacent motifs  (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition. [ 34 ] [ 124 ] [ 125 ] [ 126 ] [ 127 ] [ 128 ]  In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array. [ 129 ] [ 130 ]  The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the  leader sequence . [ 128 ] [ 131 ]


New spacers are added to a CRISPR array in a directional manner, [ 32 ]  occurring preferentially, [ 81 ] [ 124 ] [ 125 ] [ 132 ] [ 133 ]  but not exclusively, adjacent [ 127 ] [ 130 ]  to the leader sequence. Analysis of the type I-E system from  E. coli  demonstrated that the first direct repeat adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats. [ 108 ] [ 129 ]


The PAM sequence appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (nt) adjacent to the first nt of the protospacer. This nt becomes the final base in the first direct repeat. [ 109 ] [ 134 ] [ 135 ]  This suggests that the spacer acquisition machinery generates single-stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs in other organisms do not show the same level of conservation in the final position. [ 131 ]  It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition.


Analysis of  Sulfolobus solfataricus  CRISPRs revealed further complexities to the canonical model of spacer insertion, as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence. [ 130 ]


Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was discovered in the type I-E system of  E. coli . A significant enhancement in spacer acquisition was detected where spacers already target the phage, even mismatches to the protospacer. This 'priming' requires the Cas proteins involved in both acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the priming spacer. [ 109 ] [ 134 ] [ 135 ]  This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer. [ 135 ]


CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array. [ 30 ]  This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR-Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops [ 136 ] [ 137 ] [ 138 ]  created by the pairing of identical repeats that flank the crRNA. [ 139 ]  These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.


Type III systems also use Cas6, however, their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence. [ 140 ] [ 141 ] [ 142 ]


Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a  trans-activating crRNA  (tracrRNA). [ 42 ]  Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems, the crRNA does not contain the full spacer, which is instead truncated at one end. [ 95 ]


CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system. [ 7 ] [ 41 ] [ 105 ] [ 109 ] [ 143 ] [ 144 ] [ 145 ]  The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA. [ 146 ] [ 147 ]


During the interference stage in type I systems, the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits  Cas3  for DNA degradation.


Type II systems rely on a single multifunctional protein,  Cas9 , for the interference step. [ 95 ]  Cas9 requires both the crRNA and the tracrRNA to function and cleave DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is required in type II systems. However, the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).


Type III systems, like type I require six or seven Cas proteins binding to crRNAs. [ 148 ] [ 149 ]  The type III systems analysed from  S. solfataricus  and  P. furiosus  both target the mRNA of phages rather than phage DNA genome, [ 87 ] [ 149 ]  which may make these systems uniquely capable of targeting RNA-based phage genomes. [ 86 ]  Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10. [ 150 ]  The DNA cleavage was shown to be transcription dependent. [ 151 ]


The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage. [ 152 ]  RNA-guided CRISPR enzymes are classified as  type V restriction enzymes .


The cas genes in the adaptor and effector modules of the CRISPR-Cas system are believed to have evolved from two different ancestral modules. A  transposon -like element called  casposon  encoding the Cas1-like integrase and potentially other components of the adaptation module was inserted next to the ancestral effector module, which likely functioned as an independent innate immune system. [ 153 ]  The highly conserved cas1 and cas2 genes of the adaptor module evolved from the ancestral module while a variety of class 1 effector cas genes evolved from the ancestral effector module. [ 154 ]  The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events. [ 155 ]  On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements. [ 156 ]  These mobile genetic elements took the place of the multiple gene effector modules to create single gene effector modules that produce large proteins which perform all the necessary tasks of the effector module. [ 156 ]  The spacer regions of CRISPR-Cas systems are taken directly from foreign mobile genetic elements and thus their long-term evolution is hard to trace. [ 157 ]  The non-random evolution of these spacer regions has been found to be highly dependent on the environment and the particular foreign mobile genetic elements it contains. [ 158 ]


CRISPR-Cas can immunize bacteria against certain phages and thus halt transmission. For this reason,  Koonin  described CRISPR-Cas as a  Lamarckian  inheritance mechanism. [ 159 ]  However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works". [ 160 ]  But as more recent studies have been conducted, it has become apparent that the acquired spacer regions of CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are genetic mutations that are acquired and then passed on. [ 161 ]  On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution. [ 161 ]


Analysis of CRISPR sequences revealed  coevolution  of host and viral genomes. [ 162 ]


The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To resist a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts' given point mutations in the spacer. [ 152 ]  Similar stringency is required in PAM or the bacterial strain remains phage sensitive. [ 125 ] [ 152 ]


A study of 124  S. thermophilus  strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition. [ 124 ]  Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A  comparative genomic  analysis showed that  E. coli  and  S. enterica  evolve much more slowly than  S. thermophilus . The latter's strains that diverged 250,000 years ago still contained the same spacer complement. [ 163 ]


Metagenomic  analysis of two acid-mine-drainage  biofilms  showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other. [ 81 ]  In the oral cavity, a temporal study determined that 7–22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals. [ 133 ]


From the same environment, a single strain was tracked using  PCR  primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added three spacers over 17 months, [ 133 ]  suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.


CRISPRs were analysed from the metagenomes produced for the  Human Microbiome Project . [ 164 ]  Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from  streptococcal  species and contained ≈15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time. [ 164 ]


CRISPR evolution was studied in  chemostats  using  S. thermophilus  to directly examine spacer acquisition rates. In one week,  S. thermophilus  strains acquired up to three spacers when challenged with a single phage. [ 165 ]  During the same interval, the phage developed  single-nucleotide polymorphisms  that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations. [ 165 ]


Another  S. thermophilus  experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high-phage titres. [ 166 ]  The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.


CRISPRs are widely distributed among bacteria and archaea [ 91 ]  and show some sequence similarities. [ 139 ]  Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match. [ 167 ]


Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available,  polymerase chain reaction  (PCR) can be used to amplify CRISPR arrays and analyse spacer content. [ 124 ] [ 133 ] [ 168 ] [ 169 ] [ 170 ] [ 171 ]  However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase PCR primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples; amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays. [ 171 ]


The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely  de novo  identification [ 172 ]  or by using direct repeat sequences in partially assembled CRISPR arrays from  contigs  (overlapping DNA segments that together represent a consensus region of DNA) [ 164 ]  and direct repeat sequences from published genomes [ 173 ]  as a hook for identifying direct repeats in individual reads.


Another way for bacteria to defend against phage infection is by having  chromosomal islands . A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from a bacterial chromosome upon phage infection and can inhibit phage replication. [ 174 ]  PICIs are induced, excised, replicated, and finally packaged into small capsids by certain staphylococcal temperate phages. PICIs use several mechanisms to block phage reproduction. In the first mechanism, PICI-encoded Ppi differentially blocks phage maturation by binding or interacting specifically with phage TerS, hence blocking phage TerS/TerL complex formation responsible for phage DNA packaging. In the second mechanism PICI CpmAB redirects the phage capsid morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can package only 1/3rd of their genome in these small capsids and hence become nonviable phage. [ 175 ]  The third mechanism involves two proteins, PtiA and PtiB, that target the LtrC, which is responsible for the production of virion and lysis proteins. This interference mechanism is modulated by a modulatory protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and hence achieves the required level of interference. [ 176 ]


One study showed that lytic ICP1 phage, which specifically targets  Vibrio cholerae   serogroup  O1, has acquired a CRISPR-Cas system that targets a  V. cholera  PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be  homologous  to the I-F system found in  Yersinia pestis . Moreover, like the bacterial CRISPR-Cas system, ICP1 CRISPR-Cas can acquire new sequences, which allows phage and host to co-evolve. [ 177 ] [ 178 ]


Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one or two spacers. It has been shown that spacers within the virus-borne CRISPR arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays represent a mechanism of heterotypic superinfection exclusion and participate in interviral conflicts. [ 171 ]


CRISPR gene editing is a revolutionary technology that allows for precise, targeted modifications to the DNA of living organisms. Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is the most commonly used system, that allows "cutting" of DNA at specific locations and either delete, modify, or insert genetic material. This technology has transformed fields such as genetics, medicine, [ 179 ] [ 180 ]  and agriculture, [ 181 ] [ 182 ]  offering potential treatments for genetic disorders, advancements in crop engineering, and research into the fundamental workings of life. However, its ethical implications and potential unintended consequences have sparked significant debate. [ 183 ] [ 184 ]


CRISPR activation  ( CRISPRa ) is a gene regulation technique that utilizes an engineered form of the  CRISPR-Cas9  system to enhance the expression of specific genes without altering the underlying DNA sequence. Unlike traditional CRISPR-Cas9, which introduces  double-strand breaks  to edit genes, CRISPRa employs a modified, catalytically inactive  Cas9  (dCas9) fused with  transcriptional activators  to target  promoter  or  enhancer  regions, thereby boosting  gene transcription . This method allows for precise control of gene expression, making it a valuable tool for studying gene function, creating gene regulatory networks, and developing potential therapeutic interventions for a variety of diseases. [ 1 ]


Like for  CRISPR interference , the CRISPR effector is guided to the target by a complementary guide RNA. However, CRISPR activation systems are fused to transcriptional activators to increase  expression  of genes of interest. Such systems are usable for many purposes including but not limited to,  genetic screens  and overexpression of proteins of interest.
The most commonly-used effector is based on  Cas9  (from Type II systems), but other effectors like  Cas12a  (Type V) have been used as well. [ 2 ]


Cas9 Endonuclease Dead, also known as dead Cas9 or  dCas9 , is a mutant form of  Cas9  whose endonuclease activity is removed through  point mutations  in its endonuclease domains.  Similar to its unmutated form, dCas9 is used in CRISPR systems along with  gRNAs  to target specific genes or  nucleotides  complementary to the gRNA with  PAM sequences  that allow Cas9 to bind. Cas9 ordinarily has 2 endonuclease domains called the RuvC and HNH domains.  The point mutations D10A and H840A change 2 important  residues  for endonuclease activity that ultimately results in its deactivation.  Although dCas9 lacks endonuclease activity, it is still capable of binding to its guide RNA and the  DNA strand  that is being targeted because such binding is managed by other domains.  This alone is often enough to attenuate if not outright block transcription of the targeted gene if the gRNA positions dCas9 in a way that prevents transcriptional factors and RNA polymerase from accessing the DNA.  However, this ability to bind DNA can also be exploited for activation since dCas9 has modifiable regions, typically the  N  and  C terminus  of the protein, that can be used to attach transcriptional activators. [ 3 ]


See:  Guide RNA ,  CRISPR


A small guide RNA ( sgRNA ), or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets.  gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions.  The spacer region has nucleotides that are complementary to those found on the target genes, often in the  promoter  region.  The scaffold region is responsible for formation of a complex with (d)Cas9.  Together, they bind (d)Cas9 and direct it to the gene(s) of interest.  Since the spacer region of a gRNA can be modified for any potential sequence, they give CRISPR systems much more flexibility as any genes and nucleotides with a sequence complementary to the spacer region can become possible targets. [ 3 ]


See:  Transcriptional Activator ,  Transcription Factor


Transcriptional Activators  are protein domains or whole proteins linked to dCas9 or sgRNAs that assist in the recruitment of important co-factors as well as  RNA Polymerase  for  transcription  of the  gene(s)  targeted by the system. In order for a protein to be made from the gene that encodes it, RNA polymerase must make RNA from the DNA template of the gene during a process called transcription. Transcriptional activators have a DNA binding domain and a domain for activation of transcription. The activation domain can recruit general  transcription factors  or RNA polymerase to the gene sequence. Activation domains can also function by facilitating transcription by stalled RNA polymerases, and in eukaryotes can act to move  nucleosomes  on the DNA or modify  histones  to increase gene expression. [ 4 ]  These activators can be introduced into the system through attachment to dCas9 or to the sgRNA.  Some researchers have noted that the extent of transcriptional upregulation can be modulated by using multiple sites for activator attachment in one experiment and by using different variations and combinations of activators at once in a given experiment or sample. [ 5 ] [ 6 ] [ 7 ]


An  expression system  is required for the introduction of the gRNAs and (d)Cas9 proteins into the cells of interest. Typically employed options include but are not limited to  plasmids  and  viral vectors  such as  adeno-associated virus  (AAV) vector or  lentivirus  vector.


The VP64-p65-Rta, or VPR, dCas9 activator was created by modifying an existing dCas9 activator, in which a Vp64 transcriptional activator is joined to the C terminus of dCas9. [ 1 ]  In the dCas9-VPR protein, the transcription factors p65 and Rta are added to the C terminus of dCas9-Vp64. Therefore, all three transcription factors are targeted to the same gene. The use of three transcription factors, as opposed to solely Vp64, results in increased expression of targeted genes. When different genes were targeted by dCas9, they all showed significantly greater expression with dCas9-VPR than with dCas9-VP64. It has also been demonstrated that dCas9-VPR can be used to increase expression of multiple genes within the same cell by putting multiple sgRNAs into the same cell. [ 8 ]


dCas9-VPR has been used to activate the neurogenin 2 (link) and neurogenic differentiation 1 (link) genes, resulting in differentiation of  induced pluripotent stem cells  into induced  neurons . [ 8 ]  A study comparing dCas9 activators found that the VPR, SAM, and Suntag activators worked best with dCas9 to increase gene expression in a variety of  fruit fly ,  mouse , and  human  cell types. [ 9 ]


To overcome the limitation of the dCas9-VP64 gene activation system, the dCas9-SAM system was developed to incorporate multiple transcriptional factors. Utilizing  MS2 ,  p65 , and  HSF1  proteins, dCas9-SAM system recruits various transcriptional factors working synergistically to activate the gene of interest.


In order to assemble different transcriptional activators, the dCas9-SAM system uses a modified  single guide RNA (sgRNA)  that has binding sites for the MS2 protein. Hairpin  aptamers  are attached to the tetra loop and the stem loop 2 of the sgRNA to become binding sites for dimerized MS2 bacteriophage coat proteins. As the hairpins are exposed outside of the dCas9-sgRNA complex, other transcriptional factors can bind to the MS2 protein without disrupting the dCas9-sgRNA complex. Thus, the MS2 protein is engineered to include p65 and HSF1 proteins. The MS2-p65-HSF1 fusion protein interacts with the dCas9-VP64 to recruit more transcriptional factors onto the promoter of the target genes.


Employing the dCas-SAM system, Zhang et al. (2015) successfully reactivated the latent HIV gene to over-express viral proteins from the HIV host cells. [ 10 ]  They were able to over-express viral proteins substantially to trigger apoptosis of HIV-1 latent cells due to the toxicity of viral proteins. In another dCas-SAM system experiment, Konermann et al. (2015) found genes in melanoma cells that give resistance to a BRAF inhibitor through activating candidate genes via dCas system. [ 7 ]  Thus, the dCas9-SAM system can further be employed to activate latent genes, develop gene therapies, and discover new genes.


The SunTag activator system uses the dCas9 protein, which is modified to be linked with the SunTag. The SunTag is a repeating polypeptide array that can recruit multiple copies of  antibodies . Through attaching transcriptional factors on the antibodies, the SunTag dCas9 activating complex amplifies its recruitment of transcriptional factors. In order to guide the dCas9 protein to its target gene, the dCas9 SunTag system uses  sgRNA .


Tanenbaum et al.(2014) are credited for creating the dCas9 SunTag system. [ 11 ]  For the  antibodies , they employed  GCN4  antibodies which was bound to transcriptional factor VP64. In order to transport the antibodies to the nuclei of the cells, they attached  NLS  tag. To confirm the nuclear localization of the antibodies,  sfGFP  was used for visualization purpose. Therefore, the GCN4-sfGFP-NLS-VP64 protein was developed to be interact with dCas SunTag system. The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines. [ 11 ]  Comparing with the dCas9-VP64 activation complex, they were able to increase the  CXCR4  gene expression 5-25 times greater in K562 cell lines. Not only was there a greater CXCR4 protein overexpression but also CXCR4 proteins were active to further travel on the transwell migration assay. Thus, the dCas9-SunTag system can be used to activate genes that are present latently such as virus genes.


The dCas9 activation system allows a desired gene or multiple genes in the same cell to be expressed. It is possible to study genes involved in a certain process using a  genome  wide screen that involves activating expression of genes. Examining which sgRNAs yield a phenotype suggests which genes are involved in a specific pathway. The dCas9 activation system can be used to control exactly which cells are activated and at what time activation occurs. dCas9 constructs have been made that turn on a dCas9-activator fusion protein in the presence of light or chemicals. Cells can also be  reprogrammed  or differentiated from one cell type into another by increasing the expression of certain genes important for the formation or maintenance of a cell type. [ 12 ]


One research group used a system in which dCas9 was fused to a particular domain, C1B1. When blue light is shined on the cell, the  cryptochrome  2 (Cry2) domain binds to C1B1. The Cry2 domain is fused to a  transcriptional activator , so blue light targets the activator to the spot where dCas9 is bound. The use of light allows a great deal of control over when the targeted gene is activated. Removing the light from the cell results in only dCas9 remaining at the target gene, so expression is not increased. In this way, the system is reversible. [ 13 ]  A similar system was developed using chemical control. In this system, dCas9 recruits an MS2 fusion protein that contains the domain FKBP. In the presence of the chemical RAP, an FRB domain fused to a chromatin modifying complex binds to FKBP. Whenever RAP is added to the cells, a specific  chromatin modifier complex  can be targeted to the gene. That allows scientists to examine how specific  chromatin  modifications affect the expression of a gene. [ 14 ]  The dCAs9-VPR system is used as an activator by targeting it to the promoter of a gene upstream of the coding region. A study used various sgRNAs to target different portions of the gene, finding that the dCas9-VPR activator can act as an activator or a repressor, depending on the location it binds. In a cell, sgRNAs targeting the  promoter  could allow dCas9-VPR to increase expression, while sgRNAs targeting the coding region of the gene result in dCas9-VPR decreasing expression. [ 15 ]


The versatility of sgRNAs allows dCas9 activators to increase the expression of any gene within an organism's genome. That could be used to increase expression of a protein coding gene or a transcribed RNA. A paper demonstrated that genome wide activation could be used to determine which proteins are involved in mediated resistance to a specific drug. [ 7 ]  Another paper used genome wide activation of  long, noncoding RNAs  and observed that increasing the expression of certain long noncoding RNAs conferred resistance to the drug vemurafenib. [ 16 ]  In both cases, the cells that survive the drug could be studied to determine which sgRNAs they contain. That allows researchers to determine which gene was activated in each surviving cell, which suggests which genes are important for resistance to that drug.


A dCas9 fusion with VP64, p65, and  HSF1  (heat shock factor 1) allowed researchers to target genes in  Arabidopsis thaliana  and increase transcription to a similar level as when the gene itself is inserted into the plant's genome. For one of the two genes tested, the dCas9 activator changes the number and size of leaves and made the plants better able to handle drought. The authors conclude that the dCas9 activator can create phenotypes in plants that are similar to those observed when a transgene is inserted for overexpression. [ 17 ]  Researchers have used multiple guide RNAs to target dCas9 activation system to multiple genes in a specific mouse strain in which dCas9 can be turned on in specific cell lines using the  Cre recombinase  system. Scientists used the targeting and increased expression of several genes to examine the processes involved in  regeneration  and  carcinomas  of the  liver . [ 18 ]


CRISPR-Cas design tools  are computer software platforms and  bioinformatics  tools used to facilitate the design of  guide RNAs  (gRNAs) for use with the  CRISPR/Cas  gene editing system.


The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases) system was originally discovered to be an acquired immune response mechanism used by  archaea  and  bacteria . It has since been adopted for use as a tool in the  genetic engineering  of higher organisms.


Designing an appropriate gRNA is an important element of genome editing with the CRISPR/Cas system. A gRNA can and at times does have unintended interactions ("off-targets") with other locations of the genome of interest. For a given candidate gRNA, these tools report its list of potential off-targets in the genome thereby allowing the designer to evaluate its suitability prior to embarking on any experiments.


Scientists have also begun exploring the mechanics of the CRISPR/Cas system and what governs how good, or active, a gRNA is at directing the Cas nuclease to a specific location of the genome of interest. [ 1 ] [ 2 ]  As a result of this work, new methods of assessing a gRNA for its 'activity' have been published, [ 1 ] [ 2 ]  and it is now best practice to consider both the unintended interactions of a gRNA as well as the predicted activity of a gRNA at the design stage.


The below table lists available tools and their attributes.


Cas12a  ( C RISPR- as sociated protein  12a , previously known as  Cpf1 ) is an RNA-guided  endonuclease  that forms an essential component of the  CRISPR  systems found in some  bacteria  and  archaea . In its natural context, Cas12a targets and destroys the genetic material of  viruses  and other foreign  mobile genetic elements , thereby protecting the host cell from infection. Like other Cas enzymes, Cas12a binds to a "guide"  RNA  (termed a  crRNA , or  CR ISPR  RNA ) which targets it to a DNA sequence in a specific and programmable matter. In the host organism, the crRNA contains a constant region that is recognized by the Cas12a protein and a "spacer" region that is complementary to a piece of foreign nucleic acid (e.g. a portion of a  phage  genome) that previously infected the cell. [ 1 ]


As with  Cas9  and other Cas proteins, the programmable DNA-targeting activity of Cas12a makes it a useful tool for biotechnology and biological research applications. By modifying the spacer sequence in the crRNA, researchers can target Cas12a to specific DNA sequences, allowing for highly targeted  modifications of DNA . [ 2 ]  Cas12a is distinguished from Cas9 by a its single  RuvC  endonuclease active site, its 5'  protospacer adjacent motif  preference, and its formation of  sticky rather than blunt ends  at the cut site; these and other differences may make it more suitable for certain applications [ citation needed ] . Beyond its use in  basic research , CRISPR-Cas12a could have applications in the treatment of  genetic illnesses  and in implementing  gene drives . [ 2 ]


CRISPR-Cas12a was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Its identification through  bioinformatics  as a CRISPR system protein, its naming, and a  hidden Markov model  (HMM) for its detection were provided in 2012 in a release of the  TIGRFAMs  database of  protein families . Cas12a appears in many bacterial species. The ultimate Cas12a endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it. [ 3 ]  Two candidate enzymes from  Acidaminococcus  and  Lachnospiraceae  display efficient genome-editing activity in human cells. [ 2 ]


CRISPR-Cas systems are separated into two classes:  Class I , in which several Cas proteins associate with a crRNA to build a functional endonuclease, and  Class II , in which a single Cas endonuclease associates with a crRNA; Class II is further divided into  Type II ,  Type V , and  Type VI  systems. Cas12a is identified as a  Class II, Type V  CRISPR-Cas system. [ 4 ]


The acronym CRISPR ( C lustered  R egularly  I nterspaced  S hort  P alindromic  R epeats) refers to the invariant DNA sequences found in bacteria and archaea which encode Cas proteins and their crRNAs. Cas12a was originally known as  Cpf1 , an abbreviation of CRISPR and two  genera  of bacteria where it appears,  Prevotella  and  Francisella . It was renamed in 2015 after a broader rationalization of the names of Cas ( C RISPR  as sociated) proteins to correspond to their  sequence homology . [ 4 ]


The Cas12a protein contains a mixed alpha/beta domain, a  RuvC -like endonuclease domain (broken into two non-contiguous segments, RuvC-I and RuvC-II) similar to the RuvC domain of Cas9, and a  zinc finger -like domain [ 5 ] . Unlike Cas9, Cas12a does not have an HNH endonuclease domain, and the N-terminal region of Cas12a does not have an alpha-helical recognition lobe as seen in Cas9. [ 4 ]


The Cas12a loci encode  Cas1 ,  Cas2  and  Cas4  proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cas12a-family proteins in many bacterial species. [ 4 ]


Also unlike Cas9, Cas12a does not require a  tracrRNA  (which in natural CRISPR systems must base-pair with a separate crRNA before binding to a Cas protein), instead binding a single crRNA. Both Cas12a and its guide RNA are smaller than the protein and RNA components of the Cas9 system; the crRNA of Cas12a is approximately half as long as sgRNAs used with Cas9. [ 6 ]  This reduced size renders Cas12a more suitable for applications such as  in vivo  delivery via  adeno-associated virus  (AAV), which have limited DNA packaging capacity due to their small capsids.


The Cas12a-crRNA complex cleaves target DNA or RNA by identification of a  protospacer adjacent motif  (PAM) 5'-YTN-3' [ 7 ]  (where "Y" is a pyrimidine [ 8 ]  and "N" is any  nucleobase ), in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cas12a introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang. [ 5 ]


The CRISPR-Cas12a system consist of a Cas12a enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR-Cas12a systems activity has three stages: [ 3 ]


Cas9 requires two  RNA  molecules to cut DNA while Cas12a needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind  blunt ends . Cas12a leaves one strand longer than the other, creating  sticky ends . The sticky ends have different properties than blunt ends during  non-homologous end joining  or  homologous repair  of DNA, which confers certain advantages to Cas12a when attempting gene insertions, compared to Cas9. [ 3 ]  Although the CRISPR-Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. [ 1 ]  Cas12a lacks  tracrRNA , utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB. [ 6 ]


In summary, important differences between Cas12a and Cas9 systems are that Cas12a: [ 10 ]


Cas12 endonucleases ultimately likely evolved from the  TnpB  endonuclease of IS200/IS605-family transposons.  TnpB , not yet "domesticated" into the CRISPR immune system, are themselves able to perform RNA-guided cleavage using a  OmegaRNA  template system. [ 11 ]


Multiple aspects influence target efficiency and specificity when using CRISPR, including guide RNA design. Many design models and  CRISPR-Cas software tools  for optimal design of guide RNA have been developed. These include SgRNA designer, CRISPR MultiTargeter, SSFinder. [ 12 ]  In addition, commercial antibodies are available for use to detect Cas12a protein. [ 13 ]


CRISPR-Cas9 is subject to  Intellectual property  disputes while CRISPR-Cas12a does not have the same issues. [ 2 ]


CRISPR gene editing  (CRISPR, pronounced  / ˈ k r ɪ s p ə r /  (crisper), refers to a  c lustered  r egularly  i nterspaced  s hort  p alindromic  r epeats") is a  genetic engineering  technique in  molecular biology  by which the  genomes  of living organisms may be modified. It is based on a simplified version of the bacterial  CRISPR - Cas9  antiviral defense system. By delivering the Cas9  nuclease  complexed with a synthetic  guide RNA  (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed or new ones added  in vivo . [ 1 ]


The technique is considered highly significant in  biotechnology  and  medicine  as it enables editing genomes  in vivo  and is precise, cost-effective, and efficient. It can be used in the creation of new medicines,  agricultural products , and  genetically modified organisms , or as a means of controlling  pathogens  and  pests . It also offers potential in the treatment of inherited  genetic diseases  as well as diseases arising from  somatic mutations  such as cancer. However, its use in  human germline genetic modification  is highly controversial. The development of this technique earned  Jennifer Doudna  and  Emmanuelle Charpentier  the  Nobel Prize in Chemistry  in 2020. [ 2 ] [ 3 ]  The third researcher group that shared the  Kavli Prize  for the same discovery, [ 4 ]  led by  Virginijus Šikšnys , was not awarded the Nobel prize. [ 5 ] [ 6 ] [ 7 ]


Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of  DNA  to introduce the modification by one of two methods. Knock-in mutations, facilitated via  homology directed repair  (HDR), is the traditional pathway of targeted genomic editing approaches. [ 1 ]  This allows for the introduction of targeted  DNA damage and repair . HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template. [ 1 ]  This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of  non-homologous end joining  (NHEJ) or  POLQ/polymerase theta -mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption.


While  genome editing  in  eukaryotic cells  has been possible using various methods since the 1980s, the methods employed had proven to be inefficient and impractical to implement on a large scale. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing became possible. Cas9 derived from the bacterial species  Streptococcus pyogenes  has facilitated targeted genomic modification in eukaryotic cells by allowing for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands. [ 8 ]  Researcher can insert Cas9 and template RNA with ease in order to  silence  or cause  point mutations  at specific  loci . This has proven invaluable for quick and efficient  mapping  of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease that significantly reduce off-target activity have been developed. [ 9 ]


CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [ 10 ]  was the  AAAS 's choice for  Breakthrough of the Year  in 2015. [ 11 ]  Many  bioethical  concerns have been raised about the prospect of using CRISPR for  germline  editing, especially in  human embryos . [ 12 ]  In 2023, the first drug making use of CRISPR gene editing,  Casgevy , was approved for use in the United Kingdom, to cure  sickle-cell disease  and  beta thalassemia . [ 13 ] [ 14 ]  Casgevy was approved for use in the United States on December 8, 2023, by the  Food and Drug Administration . [ 15 ]


In the early 2000s, German researchers began developing  zinc finger nucleases  (ZFNs), synthetic proteins whose  DNA-binding domains  enable them to create double-stranded breaks in DNA at specific points. ZFNs have a higher precision and the advantage of being smaller than Cas9, but ZFNs are not as commonly used as CRISPR-based methods. In 2010, synthetic nucleases called  transcription activator-like effector nucleases  (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the design and creation of a custom protein for each targeted DNA sequence, which is a much more difficult and time-consuming process than that of designing guide RNAs. CRISPRs are much easier to design because the process requires synthesizing only a short RNA sequence, a procedure that is already widely used for many other molecular biology techniques (e.g. creating  oligonucleotide   primers ). [ 16 ]


Whereas methods such as  RNA interference  (RNAi) do not fully suppress gene function, CRISPR,  ZFNs , and  TALENs  provide full, irreversible  gene knockout . [ 17 ]  CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low. [ 17 ] [ 18 ] [ 19 ]


In 2005, Alexander Bolotin at the French National Institute for Agricultural Research (INRA) discovered a CRISPR locus that contained novel Cas genes, significantly one that encoded a large protein known as Cas9. [ 20 ]


In 2006, Eugene Koonin at the US National Center for Biotechnology information, NIH, proposed an explanation as to how CRISPR cascades as a bacterial immune system. [ 20 ]


In 2007, Philippe Horvath at Danisco France SAS displayed experimentally how CRISPR systems are an adaptive immune system, and integrate new phage DNA into the CRISPR array, which is how they fight off the next wave of attacking phage. [ 20 ]


In 2012, the research team led by professor  Jennifer Doudna  ( University of California, Berkeley ) and professor  Emmanuelle Charpentier  ( Umeå University ) were the first people to identify, disclose, and file a patent application for the CRISPR-Cas9 system needed to edit DNA. [ 20 ]  They also published their finding that CRISPR- Cas9  could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the  history of biology .


As of November 2013 [update] , SAGE Labs (part of  Horizon Discovery  group) had  exclusive rights  from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models. [ 21 ]  By 2015 [update] ,  Thermo Fisher Scientific  had licensed intellectual property from ToolGen to develop CRISPR reagent kits. [ 22 ]


As of December 2014 [update] ,  patent rights  to CRISPR were contested. Several companies formed to develop related drugs and research tools. [ 23 ]  As companies ramped up financing, doubts as to whether CRISPR could be quickly monetized were raised. [ 24 ]  In 2014,  Feng Zhang  of the Broad Institute of MIT and Harvard and nine others were awarded US patent number 8,697,359 [ 25 ]  over the use of CRISPR–Cas9 gene editing in eukaryotes. Although Charpentier and Doudna (referred to as CVC) were credited for the conception of CRISPR, the Broad Institute was the first to achieve a "reduction to practice" according to patent judges Sally Gardner Lane, James T. Moore and Deborah Katz. [ 26 ]


The first set of patents was awarded to the Broad team in 2015, prompting attorneys for the CVC group to request the first interference proceeding. [ 27 ]  In February 2017, the US Patent Office ruled on a  patent interference  case brought by University of California with respect to patents issued to the  Broad Institute , and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California. [ 28 ] [ 29 ] [ 30 ]


Shortly after, University of California filed an appeal of this ruling. [ 31 ] [ 32 ]  In 2019 the second interference dispute was opened. This was in response to patent applications made by CVC that required the appeals board to determine the original inventor of the technology. The USPTO ruled in March 2022 against UC, stating that the Broad Institute were first to file. The decision affected many of the licensing agreements for the CRISPR editing technology that was licensed from UC Berkeley. UC stated its intent to appeal the USPTO's ruling. [ 33 ]


In March 2017, the European Patent Office (EPO) announced its intention to allow claims for editing all types of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna, [ 34 ] [ 35 ]  and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed. [ 34 ]  As of August 2017 [update]  the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad. [ 36 ]


In July 2018, the  ECJ  ruled that  gene editing  for plants was a sub-category of  GMO foods  and therefore that the CRISPR technique would henceforth be regulated in the  European Union  by their rules and regulations for  GMOs . [ 37 ]


In February 2020, a US trial showed safe CRISPR gene editing on three cancer patients. [ 38 ]


In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field. [ 39 ] [ 40 ]  They made history as the first two women to share this award without a male contributor. [ 41 ] [ 5 ]


In June 2021, the first, small  clinical trial  of  intravenous  CRISPR gene editing in humans concluded with promising results. [ 42 ] [ 43 ]


In September 2021, the first CRISPR-edited food went on public sale in Japan. Tomatoes  were genetically modified  for around five times the normal amount of possibly calming [ 44 ]   GABA . [ 45 ]  CRISPR was first applied in tomatoes in 2014. [ 46 ]


In December 2021, it was reported that the first CRISPR-gene-edited marine animal/ seafood  and second set of CRISPR-edited food has gone on public sale in Japan: two fish of which one species grows to twice the size of natural specimens due to disruption of  leptin , which controls appetite, and the other grows to 1.2 the natural average size with the same amount of food due to disabled  myostatin , which inhibits  muscle growth . [ 47 ] [ 48 ] [ 49 ]


A 2022 study has found that knowing more about CRISPR tomatoes had a strong effect on the participants' preference. "Almost half of the 32 participants from Germany who are scientists demonstrated constant choices, while the majority showed increased willingness to buy CRISPR tomatoes, mostly non-scientists." [ 50 ] [ 51 ]


In May 2021, UC Berkeley announced their intent to auction  non-fungible tokens  of both the patent for CRISPR gene editing as well as  cancer immunotherapy . However, the university would in this case retain ownership of the patents. [ 52 ] [ 53 ]  85 % of funds gathered through the sale of the collection named  The Fourth Pillar  were to be used to finance research. [ 54 ] [ 55 ]  It sold in June 2022 for 22 Ether, which was around  US$ 54,000 at the time. [ 56 ]


In November 2023, the United Kingdom's  Medicines and Healthcare products Regulatory Agency  (MHRA) became the first in the world to approve the use of the first drug based on CRISPR gene editing, Casgevy, to treat  sickle-cell anemia  and  beta thalassemia . Casgevy, or  exagamglogene autotemcel , directly acts on the genes of the stem cells inside the patient's bones, having them produce healthy red blood cells. This treatment thus avoids the need for regular, costly blood transfusions. [ 13 ] [ 14 ]


In December 2023, the  FDA  approved the first gene therapy in the US to treat patients with Sickle Cell Disease (SCD). The FDA approved two milestone treatments, Casgevy and Lyfgenia, representing the first cell-based gene therapies for the treatment of SCD. [ 57 ]


CRISPR-Cas9 genome editing uses with a  Type II  CRISPR system. This system includes a  ribonucleoprotein  (RNP), consisting of  Cas9 , crRNA, and tracrRNA, along with an optional DNA repair template.


CRISPR-Cas9  often employs  plasmids  that code for the RNP components to  transfect  the target cells, or the RNP is assembled before addition to the cells via nucleofection. [ 58 ]  The main components of this plasmid are displayed in the image and listed in the table. The crRNA is uniquely designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to specific sequences within the host cell's DNA. The crRNA must bind only where editing is desired. The repair template is also uniquely designed for each application, as it must complement to some degree the DNA sequences on either side of the cut and also contain whatever sequence is desired for insertion into the host genome.


Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). [ 59 ]  This sgRNA can be included alongside the gene that codes for the Cas9 protein and made into a plasmid in order to be transfected into cells. Many online tools are available to aid in designing effective sgRNA sequences. [ 60 ] [ 61 ]


Alternative proteins to Cas9 include the following:


CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the  protospacer adjacent motif  (PAM) sequence. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array. [ 58 ]  A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently  synthesized . [ 75 ] [ 76 ]


The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However, this is ultimately not too limiting, as it is typically a very short and nonspecific sequence that occurs frequently at many places throughout the genome (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs). [ 58 ]


Once these sequences have been assembled into a plasmid and transfected into cells, the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and – depending on the Cas9 variant – creates a single- or double-stranded break at the appropriate location in the DNA. [ 77 ]


Properly spaced single-stranded breaks in the host DNA can trigger  homology directed repair , which is less error-prone than the  non-homologous end joining  that typically follows a double-stranded break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9-induced DNA break. [ 58 ]  The goal is for the cell's native HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells. Combined transient inhibition of NHEJ and TMEJ by a small molecule and siRNAs can increase HDR efficiency to up to 93% and simultaneously prevent off-target editing. [ 78 ]