A “CRISPR” OVERVIEW OF GENOME EDITING: POTENTIALS AND ... Art_A_Crisp_Overvi… · Cas9 tool is the most versatile, simplest and precise method of genome editing and is therefore

210 SCIENCE AND CULTURE, JULY-AUGUST, 2017

A “CRISPR” OVERVIEW OF GENOME EDITING:POTENTIALS AND CHALLENGES

SUDIPTA CHAKRABORTY* AND MOULINATH ACHARYA*†

“CRISPR” stands for Clustered Regularly Interspaced Short Palindromic Repeats, which is thecharacteristic of a bacterial immune system that forms the basis for the popular CRISPR-Cas9genome editing technology. It is robust, faster, cheaper and more accurate than previous DNAediting techniques such as meganucleases, zinc finger nucleases (ZFN), and transcription activatorlike effector nucleases (TALEN). This technology has a wide range of potential applications rangingfrom basic research, agriculture, biomedicine, generating germline animal models, somatic genomeengineering , functional genome screening and in treating genetic diseases. Nowadays CRISPR-Cas9 tool is the most versatile, simplest and precise method of genome editing and is thereforeattracting a lot of attention in the scientific world. This review highlights evolution, classification,components and applications of CRISPR based technology as well as its future challenges.

ARTICLE

* National Institute of Biomedical Genomics, Kalyani, West Bengal,India 741251

† Address correspondence to: email: [email protected]

Introduction

“SEEK AND DESTROY” – we could not get thebetter compliment for the CRISPR-Cas system in terms ofgenome editing. Genome editing is a modern route ofgenetic engineering in which the target DNA can beinserted, deleted or rather be replaced in the genome of aliving organism. There are several methods have beendeveloped in the course of evolution of the genome editingtechnology. The commonality of all these methods lies incleaving the genome using “molecular scissors” in the formof innumerable nucleases of different types and origin.Another basic prerequisite of genome editing relies uponrecognizing the target of interest(s) to make the precisecut at a specific site in the genome. These nucleases areapplied to create double-strand breaks (DSBs) at desiredlocations in the gene that would be repaired either by non-homologous end-joining (NHEJ) or homologousrecombination (HR), resulting in targeted edit by creatingor correcting mutations.

During these phases of discovery, there are mostlyfour families of engineered nucleases have been used, suchas, meganucleases, zinc finger nucleases (ZFNs),transcription activator-like effector-based nucleases(TALEN), and the CRISPR-Cas system1-3 Among these,the latest discovery is, CRISPR-Cas system that has beenwidely accepted worldwide quite rapidly. The CRISPR-Castechnology has been selected as “2015 Breakthrough ofthe Year” by the renowned Science magazine. The utmostreason for gaining enormous popularity within a short timeof CRISPR lies in the maximum accuracy compared to itspredecessors such as meganucleases, ZFN or TALEN. Also,CRISPR is the only genome editing system which is guidedby RNA while the rest are mostly protein guided DNAediting system. The RNA-DNA hybrids are conformation-wise way stronger and more stable than protein-DNAhybrids. This CRISPR-Cas based Genome editing is oneof the finest techniques that molecular biology and syntheticbiology have afforded to allow deliberate influence over awide range of model organisms used in biomedicalresearch. It is characterized by its level of precision withwhich it may be targeted, and moreover its controllability.In 2012, it was discovered that a system of defense against

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viral attack found in the bacterium Streptococcus pyogenescould be adopted as a programmable system for genomeediting. The system comprises two elements. The first is‘clustered regularly interspaced short palindromic repeat’(CRISPR) RNA; and the second is ‘CRISPR-associatedprotein 9’ (Cas9), an endonuclease by nature, FokI is abacterial type IIS restriction endonuclease having of an N-terminal DNA-binding domain and a non-specific DNAcleavage domain at its C-terminal4 end. FokI domains havesite-specific nucleases which form a dimer that activatesthe nuclease activity, thus creating a DSB near their bindingsites. CRISPR are the segments of prokaryotic DNAcontaining short, repetitive base sequences. Each repetitionis followed by short segments of spacer DNA to foreignDNA5 The prototypical CRISPR components from S.pyogenes comprise two types of RNA molecule thatscientists combined into one, called a single guide RNA(sgRNA) or guide RNA (gRNA). There RNAs are beingguided to search the target region followed by recruitingprotein Cas9 for cleavage. The diagram of CRISPR locusand engineered Cas9 nuclease with its domain are depictedin the Fig: 1.

Evolution of CRISPR-Cas9 system

The study of CRISPR–Cas systems was first reported

30 years ago in 1987, during the sequencing of the geneencoding alkaline phosphatase isozyme conversion enzyme(ICE) in the Escherichia coli genome. It was found as anarray of short, repetitive DNA sequences (~20–40 bp inlength, termed “repeats”) that are inter-spaced with non-repetitive sequences termed as “spacers”6. The function ofthese spacer sequences remained elusive for a fairly longtime, until in 2005 extensive computational analysesrevealed that these repetitive arrays were present in severalbacteria and archaea. The computational analyses furtherrevealed that these repetitive arrays were found in severalbacteria and archaea and, remarkably, that the spacers wereidentical to many sequences which is present in plasmids,transposons and bacteriophages7,8. Following the discovery,these arrays, coined as ‘clustered regularly interspaced shortpalindromic repeat’ (CRISPR) were associated with a setof Cas genes9. Subsequent studies proved that Cas geneshad sequence similarity to endonuclease and helicase genefamilies encoding other nucleic acid binding proteins9-11.It was then postulated that the spacers identical to mobilegenetic elements with CRISPR–Cas systems that may actas a form of RNA-directed interference against foreigngenetic elements11. This hypothesis contributed to the firstdirect evidence that CRISPR sequences and the associatedCas proteins directed interference against bacteriophage

infection12. After that it had beenfound that the new spacersequences are naturally acquiredinto the CRISPR array subsequentto bacteriophage infection, aprotective mechanism of adaptiveimmunity in prokaryotes12-16. Overthe last decades, the mechanism ofRNA-directed interference byCRISPR–Cas systems has beenlargely explored17-19. In short,CRISPR-mediated interferenceoccurs in three stages: at firstspacer acquisition followed bycrRNA transcription andmaturation, and ultimately targetidentification followed by cleavageof the targeted region. Theevolution of CRISPR–Cas biologyis a continuous process thatexpands extremely rapid.Researchers in this field haveelegantly unveiled not only themolecular function of CRISPR–Cas systems in defense againstforeign DNA/RNA12-14,20-23 but

Figure 1: Left: A general diagram of a CRISPR locus, which has three major components: cas genes,a leader sequence, and a repeat-spacer array. Repeats are shown as Blue boxes and spacers are colouredbars. Moreover, several CRISPRs with similar sequences can be present in a single genome, only oneof which is associated with cas genes. Right: Cas9 nuclease has been engineered (from Streptococcuspyogenes) requiring only two components: the Cas9 protein and a short ~100 nucleotide guide RNA(gRNA) that form a complex that can recognize and cleave a 20 bp double-stranded DNA target site(e.g., protospacer DNA) which is complementary to the 52 end of the gRNA and is adjacent to aprotospacer adjacent motif (PAM) of 52 -NGG-32 , in which N can be any nucleotide. The singlegRNA transcript is an engineered fusion of naturally occurring CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA)67. The tracrRNA was discovered by differential RNA sequencing andproved to be an essential component for CRISPR interference in Streptococcus pyogenes bacteria91

CRISPR. The Cas9 mediated target specificity by nucleic acid interactions between the 20 nucleotidesat the 52 end of the gRNA and the protospacer DNA, further by protein–DNA interactions betweenCas9 protein and the PAM. Upon recognition of a PAM sequence, Cas9 commences sequentialunwinding of the protospacer target-site duplex, stabilized by the development of an R-loop structurebetween the protospacer DNA and the gRNA92. The complementarity of RNA–DNA between the gRNAand the target DNA strand triggers a conformational change in Cas9 which activates the cleavage ofthe target DNA strand by the HNH nuclease domain, and of the non-target strand by having its RuvCdomain92.


revealed inkling about the evolutionof these systems24-27 and theirfunctions in other biologicalprocesses28-31. This foundationalwork has eventually led to thediscovery of how this CRISPR-Cas9 can be engineered to apply inmultiple molecular biology andsynthetic biology applicationspertaining to genome editing,regulation and efficient design toinvestigate epigenetic modificationsthroughout genome. From 2005onwards, the rate of publication ofCRISPR-Cas is shown in Fig: 2.

Classification of CRISPRSystem

CRISPR–Cas systems are of three types (Type I, II,and III) according to presence of distinctive Cas proteins,encoded adjacent to the CRISPR array27. Although havingtheir conserved role in prokaryotic adaptive immunity, theyare highly diverse27,32,33. The mode of immunity is the mostconserved between the three CRISPR–Cas subtypes, whichare encoding the different Cas proteins involved in thisprocess34. These two nucleases are both necessary andsufficient for spacer acquisition, nevertheless, superfluousfor target interference35-39. Cas1 and Cas2 proteins interactfor recognizing the DNA secondary structure of the

CRISPR repeat sequence during integration of new spacers.There are several forms of CRISPR–Cas systems indicatethat Cas1 and Cas2 form complexes with Casproteins involved in target detection and mediatescleavage35,36,40-43. The types of CRISPR–Cas systems differaccording to their target identification, crRNA maturation,and interference stages of immunity. Especially, Type I andType III systems use large, multimeric protein complexesfor these activities, while the Type II systems impose asingle protein for these diverse functions42. Type I systemsuse the endonuclease Cas6 or Cas5d to cleave the CRISPR

TABLE 1: Comparison chart for three major types of CRISPR system

Type I Type II Type III

Distribution Present in both bacteria and Present exclusively in bacteria More commonly present in archaea,archaea although it is also found in bacteria

Signature Protein Cas3 Cas 9 Cas10

Types Six types Three types Two types

Protospacer Adjacent Phage or plasmid genomes that were excised as spacers also called Type III does not require PAMMotif’s (PAM) as protospacers were found adjacent to protospacer adjacent motif sequence during acquisition.

(PAM). PAM is a conserved region typically only 2 – 5 nt longand occurs within 1 – 4 bp of the protospacer sequence on eitherside depending on the system. PAMs to be important for Type Iand II systems during acquisition.

Interference In Type I systems, the PAM Type II systems rely on a single Type III systems require six or sevensequence is recognized on the multifunctional protein Cas 9, Cas proteins binding to crRNAs.crRNA complementary strand. functional crRNA and tra-crRNA. Cas 10 which signature RAMPCorrect base pairing between the PAM is recognized on the same protein which is likely involved incrRNA and protospacer signals strand as the crRNA. Cas 9 then target DNA cleavage. Two typesa conformational change in cleaves DNA using its dual HNH were found in Type III system -Cascade that activates Cas 3 for and Ruvc / RNase H-like Type III A and Type III B. Type IIIDNA degradation. endonuclease domain. A targets mRNA whereas Type III B

targets DNA

Figure 2: Number of publications on PubMed (maintained by the National Center for BiotechnologyInformation, National Institute of Health, USA) in CRISPR/Cas9 mediated genome editing per yearstarting from 2005. An almost exponential rise is observed in this graph after 2012, indicating towardsan exceptional increase in the number of publications on CRSPR-Cas9.

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array transcript within the repeat sequences which isflanking with each spacer, resulting in a short 52 repeat-derived sequence and a 32 hairpin, including a repeat-derived sequence44-51. The Cas6 protein then transports themature crRNA to a complex of Cas proteins called Cascadewhich is CRISPR-associated complex for antiviral defensehaving functions in interference by attached to thecrRNA13,47,52-58. With the formation of differentstoichiometric structure, the Type I systems form aninterference complex of five distinct Cas proteins13,42,

47,49,51. In Type I systems crRNA binds to its target DNAby recruiting the Cas3 endonuclease, which mediatestargeted degradation16,47,59. Resembling the Type I systems,Type III systems also employ Cas6 for crRNA processingby forming multiprotein without binding to the guide RNAcomplexes for target interference50. Furthermore, the Casproteins in the Type III complexes are different as the otherCRISPR systems15,60. Cas10 being a component of TypeIII interference complexes plays an important role in thesesystems, although its function has not yet been understoodproperly27. Fascinatingly, both Type III-A and III-B systemsare competent of targeting DNA and RNA22,61,62. Types Iand II systems recognize specific nucleotide sequenceadjacent to the target region but on the complementarystrand of DNA, named as PAM (protospacer adjacentmotif)5,7,63,64. By recognizing PAM, it facilitates Casinterference complex binding, DNA melting, and RNA:DNA heteroduplex formation and prevents self-targeting ofsimilar or identical sequences lacking this PAM5. Table: 1describes the three types of CRISPR systems in details.

Components of CRISPR-Cas Machinery

I. The Endonuclease : CRISPR/Cas systems havedifferent types of enzymes liable for processing foreignDNA over and above the RNA guided endonuclease forexecuting its function, when used for genome editing; theonly CRISPR protein required is the Cas9 endonuclease28.CRISPR praotein made up of three components essentialto:

i. Binds to a Guide RNA : The guide RNA facilitatesCas9 to recognize a specific genomic locus followed bycleavage on the recognition site by Cas9 and gRNAcomplex.

ii. Attaches to Target DNA in the presence of a GuideRNA leave that Upstream (5’) of a Protospacer AdjacentMotif (PAM) : Cas9 endonuclease binds to the targetedgenomic locus which is interceded together by the targetsequence contained among the guide RNA and a 3-basepair sequence known as the Protospacer Adjacent Motif orPAM. In turn for dsDNA to be cut by Cas9 and it must

have a PAM sequence straight away downstream (3’) ofthe site targeted by the guide RNA. In the absence of eitherthe guide RNA or a PAM sequence, Cas9 will neither bindnor cut the targeted locus.

iii. Cleaves Target DNA Resulting in a Double-StrandBreak (DSB) : Cas9 have two endonuclease domains: then-terminal RuvC-like nuclease domain and the HNH-likenuclease domain in the vicinity of the centre of the protein.Upon binding, Cas9 undergoes a conformational changethat positions the nuclease domains to cleave oppositestrands of the target DNA. Therefore, the concluding resultof Cas9-mediated DNA damage is a DSB within the targetDNA having ~3-4 nucleotides upstream of the PAMsequence.

II. The Synthetic Guide RNA or gRNA (sgRNA) :In the native Type II CRISPR/Cas system, Cas9 is guidedto its target sites with the assistance of two RNAs: thecrRNA which defines the genomic target for Cas9, and thetracrRNA which acts as a scaffold linking the crRNA toCas9. In CRISPR-mediated genome editing, these two smallRNAs have been condensed into one RNA sequence knownas the guide RNA (gRNA) or single guide RNA (sgRNA).The gRNA made up of both the 20 nucleotide targetsequence to direct Cas9 to a specific genomic locus andthe scaffolding sequence needed for Cas9 binding. Amodular scaffold RNA (scRNA) encodes, within a singlemolecule, where the information is specifying the targetsite in the genome by which a particular regulatory functionto be executed at that site.65 The overall schematic diagramregarding mechanism of action of CRISPR is representedin Fig: 3.

Recombination Pathway

I. The Non-Homologous End Joining (NHEJ)Repair Pathway : The NHEJ repair pathway is an errorprone repair pathway employed to repair double strandedbreaks in the absence of a suitable repair template. TheNHEJ pathway endeavor to ligate the cleaved ends of aDSB together following insertion or deletion (InDels)mutations at the DSB site, resulting frame shifts or bydisrupting the open reading frame of the targeted genethrough introducing pre-mature stop codons . Nevertheless,the Indel outcome by NHEJ remains largely random. Itcan be ensured that the maximum gene disruption bytargeting the gRNA towards the N-terminal of the gene ofinterest. This will make sure that the frame-shift mutationsdo not lead to partially functional gene product. For thatreason the gRNA has to designed in the first or secondexonic and intronic regions of the targeted gene should beavoided while adopting the NHEJ repair pathway66.


exploited to introduce specificnucleotide modifications togenomic DNA. Here, a DNA repairtemplate that has a high degree ofhomology to the sequenceimmediately upstream anddownstream of the intended editingsite is introduced along with theappropriate gRNA and Cas9nuclease. In presence of thissuitable template, the less error-prone HDR mechanism canfaithfully make the desired changesto the Cas9 induced DSB sitethrough recombination. Whendesigning the repair template, oneshould ensure that either the targetsequence is not immediatelyfollowed by the PAM sequence orthat the PAM sequence is eitherexcluded or mutated. This is toavoid the degradation of the repairtemplate by the same CRISPR Cas9system. These two repair pathwaysare provided in the Fig: 4.

Use and delivery of Cas9

Three methods have beenadopted sequentially in terms ofimplementing the delivery of Cas9.Those methods are followingbelow:

I. Designing Single GuideRNAs : The designing of sgRNAis the foremost step and it dependson conforming to a certain set ofdesigning rules. There are severalonline tools are available fordesigning sgRNA such asCRISPRScan, ChopChop andCRISPOR etc. All sgRNAs must beadjacent to a PAM site: Sp Cas9(Streptococcus pyogenes) usesNGG or a less efficient NAG67,68.Sa Cas9’s (Staphylococcus aureus)PAM is NNGRRT. A sgRNAexpressed from a U6 promoter in

mammalian cells that should not have a stretch of four ormore uracils (U’s) in a row or rather it would be terminatedprematurely due to the activity of RNA polymerase III69.

Figure 3: The mechanism of action CRISPR-Cas9 system. A) in bacterial genomes CRISPR arraysare transcribed into pre-crRNAs containing both the spacer region and the direct repeat region. B)Cas9, RNaseIII, and the tracrRNA, bind to these transcripts and C) cleavage is done leaving maturecrRNAs bound to the Cas9/tracrRNA complex. D) The mature crRNA is needed to guide the Cas9complex to the target DNA which is E) cleaved leaving a F) double-strand break. A “gRNA” is adesigned hybrid of the tracrRNA and the crRNA. The “direct repeat region” combined with the tracrRNAforms the scaffold portion of a gRNA and the “spacer region” forms the target sequence.

II. The Homology Directed Repair (HDR) withCas9 Nuclease : The Homology Directed Repair (HDR)pathway is a more precise repair mechanism can be

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A stretch of U’s near the 3´ end of the guide sequence ishostile for Cas9–sgRNA binding31. Moreover, containingthe long stretches of the same nucleotide greatly decreasessgRNA activity70. The 5’ end of sgRNAs that append a G(guanine) is strongly recommended for high expressionfrom a U6 promoter. In addition, G is also preferred in

the first or second position closestto PAM, which may help inattachment of Cas9. A (adenine) ispreferred in the middle of sgRNAand G is preferred in the PAM-distal region whereas C (cytosine)is strongly unfavorable in the samepositions.

II. Choosing Target Sites :CRISPR interference is mosteffective with sgRNAs in the “50to +300 bp window around thetargeted site and CRISPR activationare targeted to a window between“400 to “50 bp upstream of theTargeted sequence site70.

III. Delivery Methods :There are several methods havebeen adopted regarding delivery of

vectors. After picking an appropriate Cas9-encoding vector,a complementary sgRNA-encoding vector can also beadopted. This can be done using lentiviral or retroviralvectors. This method cannot be preferred for primary celllines where it is possible to use vectors that encode bothCas9 and a single sgRNA71,72.

Cas Proteins are theBackbone of CRISPRMediated Genome Editing

CRISPR–Cas systems havebeen emerged as one of the mostexciting fields in biology having itsunique genetic elements forfunctioning as a Prokaryoticadaptive immune system. CRISPR-Cas9 mediated system has beenused throughout multiple fields likeclinical biotechnology, translationalmedicine and is rapidly changingthe direction of eukaryotic genetics.Applications of CRISPR Cas9 wasfirst to be found as a genomeediting tool in human cell culturein 201267. It has been successfullyused in multiple eukaryotic animalmodels such as baker’s yeast73,plants74, zebra fish75, fruit flies76,nematodes77, mice78, and a numberof other organisms. CRISPR-Cas9is also used as a therapeutic agent

Figure 4: Cas9 nuclease can produce either blunt or 1 bp 52 -staggered ends. Cas9 nuclease-induced double-stranded breaks (DSBs) can be repaired by one of two competing DNA repair pathwaysin mammalian cells. First, the error–prone non-homologous end-joining (NHEJ) pathway, results infrequent insertions or deletions (indels) that are often exploited to create knockout or frame-shiftmutations. The second type of DSB is the more precise homology-directed repair (HDR), which in thepresence of a user-supplied donor template used for gene correction or knock-in experiments.

Figure 5: Applications of CRISPR system. The CRISPR is extensively used by several areas of LifeSciences and allied fields starting from pharmacogenomics, differentially targeted protein engineeringpertinent to drug design, in ecological vector control and viral gene disruption. The requirement ofusing CRISPR-Cas9 mediated genome editing is increasing every day in plant sciences, specifically incrop production. In biomedical sciences, efforts are being carried out in several human diseases startingfrom monogenic rare disorders to multifactorial chronic and more complex diseases.


for curing genetic disorders and viral infection. The gRNAhas been targeted in the long-terminal repeat promoter ofthe HIV-1 genome to notably repress its expression ininfected human cells79. This system can be used inbioengineering applications, with their function being aprotective system against nucleic acids in microbialphysiology. The variants of Cas9 will allow researchers tofurther understand the structural and sequence requirementsthat determine specificity of PAM, requirements of crRNAsequence, and DNA binding stringency, allowing Cas9proteins to be engineered for increased specificity andefficacy.

Using CRISPR in Epigenomic Editing

There are some recent studies have reported the useof dCas9 systems to achieve site-specific epigenomeediting80-82. The Nm (Neisseria meningitides) dCas9 wasfused with the histone demethylase LSD1 for targeting theenhancer of genes such as Oct4, Tbx3 to maintainpluripotency in mouse embryonic stem cells (mESCs)80.Fused Nm dCas9-LSD1 efficiently suppressed theexpression of genes controlled by the targeted enhancers,and decreasing the level of the epigenetic marks H3K4me2and H3K27ac near the targeted Tbx3 enhancer region, andalso caused changes in cell morphology. Another studyshowed that the dCas9-KRAB fusion has been inducedH3K9 trimethylation (H3K9me3) when targeted to the HS2enhancer and concomitantly suppress the expression ofglobin genes that is also regulated by the HS2 enhancers82.

Use of CRISPR/Cas9 to Generate AnimalModels

Animal models have been used extensively forCRISPR to investigate neurological disorders and to findtherapeutic targets. Several neurodegenerative diseasescaused by genetic mutations and the ability of CRISPR/Cas9 to directly target any gene in one or two alleles ofthe embryonic genome opens up a new avenue for usingthis new technology to generate animal models ofneurodegenerative diseases. The traditional gene targetingtechnology made it difficult to establish large animal modelsof human diseases due to the lack of embryonic stem celllines. Since large animals are closer to humans, theirdisease models may more faithfully mimic the clinicalsymptoms of patients and are important for exploring themechanisms and treatment of both neuropsychiatricdisorders and age-related neurodegenerative diseases. Forexample, it is well known that the loss of function of theParkin and Pink1 genes cause PD. CRISPR/Cas9-mediatedmutations can mimic knockout of the Parkin and/or Pink1

gene83. CRISPR/Cas9 was found to disrupt the functionalityof dystrophin gene in monkeys and causes the same muscleatrophy phenotype seen in Human patients84.

Live Imaging of the Cellular Genome

To visualize DNA in living cells requires a robustmethod. Traditional method includes fluorescence in situhybridization (FISH) which requires fixation of a sampleand thus unable to study live processes. Inactive Cas9 (dCas9) can be fused with fluorescent protein such as EGFPand this fluorescently tagged Cas9 labeling of specific DNAloci was a powerful live cell imaging technique alternativeto FISH[85] This technique has been expanded tomulticolor and multilocus imaging for studying complexchromosomal architecture and nuclear organization.

CASFISH assays has colored dCas9/sgRNAcomplexes that allow multicolor labeling ( by fluorescentdye) of target loci in cells. In addition, the CASFISH assayis remarkably rapid under optimal conditions and isapplicable for detection in primary tissue sections. It is arobust and cost-effective technology adding a valuable toolfor basic research and genetic diagnosis86. The Summarizedfeatures of CRISPR technology are shown in Table 2.

TABLE 2: Summarized Features of CRISPR-CasTechnology

Feature CRISPR-Cas Technology

Mode of action Modifies gene via Knockout/knock in

Targeting Sites adjacent to PAM

Utilizes the endogenous DSB repair systems ( HDR andmachinery NHEJ)

Site Nucleus

Duration of effect Permanent and heritable changes

Efficiency 10% -40% editing per allele

Clonal isolation Usually Requires

Phenotype effect May not be detectable in a cellpopulation

Future Directions and Challenges

In spite of advancement of CRISPR-Cas technologyover past few years, there are still plenty questions remainunanswered that would lead to the development of morecost-effective and accurate tools in the field of genomeengineering. By now, several Cas proteins have beenpredicted to have various and conserved functions, forexample, Cas1 and Cas2 have been anticipated as a toxin–antitoxin system, turning autotoxic in the presence ofbacteriophage infections that are not successfully controlled

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by the nucleic acid targeting activity of CRISPR–Cassystems, probably by cleaving endogenous mRNA87. Thissecond line of defense would prevent bacteriophages fromreplicating and consequently infecting other cells but, iftrue, could also form the platform for a Cas2-based RNAinterference technology. Cpf1 is an RNA-guidedendonuclease of a class II CRISPR/Cas system. This typeof acquired immune mechanism is found in Prevotella andFrancisella bacteria.Cpf1 genes are associated with theCRISPR locus, coding for an endonuclease that use a guideRNA to find and cleave viral DNA .Cpf1 is a smaller andsimpler endonuclease than Cas9, overcoming some of theCRISPR/Cas9 system limitations since Cas9 requires twoRNA molecules [crRNA+ tracrRNA = gRNA] to cut DNAwhile Cpf1 requires only one. Cpf1 thus makes thetracrRNA redundant for its action, becoming a T-rich PAMand cleaves DNA via a staggered DNA double strand break.Cpf1 cuts DNA at different places that leading toresearchers to have more options while selecting an editingsite. Cas9 cuts the both strands of DNA molecule at thesame position, creating ‘blunt’ ends while Cpf1 leaves onestrand longer than the other, creating ‘sticky’ ends whichare easier to work on. Several studies have shown that theCpf1 came out to be more efficient to insert new sequencesat the cut site, contrast to Cas9.88. Concurrent study figuredout that how Cas1 and Cas2 act to incorporate newsequences into the bacterial chromosome which may furtherallow the generation of innovative technologies that aremore efficacious in mediating the site-directed DNAincorporation.

A nuclease deficient “dCas9” is developed, havingmutated nuclease domains of Cas9 from Streptococcuspyogenes (by making an H840A mutation in the HNHdomain and a D10A mutation in the RuvC domain)89.Although this “blunt” and “dead” version of Cas9 is nolonger be able to cleave DNA, but it can still target andbind DNA with the same precision when guided by thesgRNA. However, instead of irreversibly altering thegenome, binding of dCas9 interferes with the transcriptionof the target site—resulting in the reversible silencing ofthe gene. The use of dCas9 itself as a method oftranscriptional interference is only the beginning. Soon afterthe attaching effectors like repressor proteins or activatordomains, it becomes handy for reversible gene activation,epigenetic editing, and much more. Whether it is a promoterregion, regulatory region, or coding region, researcherscould use the CRISPR-dCas9 system as a modular scaffoldfor easy effectors attachment, enabling the control of anygene without introducing irreversible DNA-damagingmutations. Thus, these proteins can be programmed torecognize specific sequences of DNA. Not only that the

CRISPR-Cas system has already provided a new arena tothe modern biology for its easy application but furtherenhancement of knowledge about the functions of differentCas proteins will help to develop novel molecular toolsfor resolving many biological enigma, which otherwiseremain elusive. The schematic diagram of uses of CRISPR-Cas technology is represented in Fig: 5.

There are still several challenges in terms of successfulimplementation of CRISPR-Cas system. First of all, thereis a risk of incomplete or rather inaccurate editing thateffects mosaicism or may be off-target effects orirreversibility of genetic edits. Moreover, a number ofethical issues are also being considered such as, a potentialfor permanent genetic enhancements which affects the socialstructure, “designer babies”, feeling of otherness, potentialconflicts with religion i.e., the idea of “playing God” andmoreover, the moral considerations of altering evolution.Despite that, the risks and ethical concerns are less inbiomedical research that utilizes somatic (non-reproductive)cells as a model the CRISPR-Cas9 technique provides anadequate foundation for rapidly moving the methodologyinto clinical settings. On the contrary, editing germline cellsopens up an entirely different set of unknowns and ethicaldilemma. Germline editing has a great claim in which itwould seemingly harness the ability to “correct” geneticmutations present in an individual’s genes before thosegenes are passed on to the next offspring, with the promiseof eradicating heritable conditions or diseases. Not only isit impossible to know some of the ways in which viableoffspring might be affected, but the limitations of currenttechnologies would necessitate selective abortion while theediting has not been successful. A second editingentanglement is applied to somatic cells which revolvearound the notion of enhancement as opposed to correctinga detrimental mutation. However, the somatic enhancementmight have therapeutic value for an individual traits orcapacities through germline editing hikes several questionson eugenics and also undesirable repercussions, forinstance, the societal treatment of individuals withdisabilities to unfair access to such type of technology onthe basis of socioeconomic status. Furthermore, despite thegreat potential of CRISPR-Cas system in research andtherapeutics, improvements can still be made in itsspecificity, efficiency, and spatiotemporal control90. As aresult, we have not been able to yet harness the fullpotential of the CRISPR-Cas system. This technology hasbrought forth revolutionary changes in genomic research,together with genome editing, regulation and imaging. Thisgroundbreaking technology may continue to surprise us inthe future with its more elegant mechanisms and function,


offering the most powerful tool to move forward into anovel and unique post-genomic era.

GLOSSARY

CRISPR: Clustered Regularly Interspaced ShortPalindromic Repeat, a region in bacterial genomes used inpathogen defense.

Cas: CRISPR Associated Protein, the Cas9 nucleaseis the active enzyme for the Type II CRISPR system.

CRISPR/Cas (CRISPR-associated) systems:Clustered Regulatory Interspaced Short PalindromicRepeats are loci that have multiple short repeats providingacquired immunity to bacteria and archaea from the virus.It depends on crRNA and tracrRNA for sequence-specificsilencing of invading foreign DNA. This type II CRISPR/Cas systems, Cas9 provide as an RNA-guided DNAendonuclease that cleaves DNA upon crRNA–tracrRNAtarget recognition.

RNAi: RNAi is the mechanism through which RNAinhibit or silence gene expression.

crRNA: CRISPR RNA base pairs with tracrRNA forguiding the Cas9 endonuclease to complementary DNAsites for cleavage.

DSB: double-strand breaks are a form of DNAdamage that occurs when both DNA strands are cleaved;it is the product of ZFN, TALEN, and CRISPR/Cas9 action.

HDR: homology-directed repair is a template-dependent pathway for repair double stranded DNA lesions.

NHEJ: nonhomologous end joining is categorized asa DSB repair pathway that joins two broken ends togetherwithout the need of homologous template leading to theintroduction of small insertions and deletions at the site ofthe break to that knockout gene function.

PAM: Protospacer Adjacent Motifs are shortnucleotide motifs (2-6 base pair) that appear on crRNAand are specifically targeted by Cas9 for DNA cleavage.

tracrRNA: trans-activating chimeric RNA isnoncoding RNA that promotes crRNA processing and isneeded for activating RNA-guided cleavage by Cas9.

sgRNA: single Guide RNA, a synthetic fusion of theendogenous bacterial crRNA and tracrRNA; gives bothtargeting specificity and the binding ability for Cas9nuclease.

InDel: Insertion or Deletion, a type of mutation thatcan result in the disruption of a gene by transferring theOpen Reading Frame or by creating premature stop codons.

TALENs: transcription activator-like effectornucleases are an amalgam of the FokI cleavage domainand DNA-binding domains obtained from TALE proteins.TALENs generated DSBs that activate DNA damageresponse pathways facilitating custom alterations.

ZFNs: zinc-finger nucleases are an amalgam of thenonspecific DNA cleavage domain from the FokI restrictionendonuclease with zinc-finger proteins. ZFN dimers activatetargeted DNA DSBs that stimulate DNA damage responsepathways.

dCas9: Nuclease dead Cas9, an enzymatically inactiveform of Cas9; Can bind, but cannot cleave DNA.

Acknowledgements

This work is supported by NIBMG intramuralfunding. The authors are indebted to Prof. SharmilaSengupta, Director, NIBMG and Prof. Partha PratimMajumdar, Founder Director and Distinguished Professor,NIBMG, for their constant support and encouragement.

References1. V. M. Bedell, et al., In vivo genome editing using a high-

efficiency TALEN system. Nature, 491(7422): p. 114-8, (2012).2. K. M. Esvelt, et al., Orthogonal Cas9 proteins for RNA-guided

gene regulation and editing. Nat Methods, 10(11): p. 1116-21,(2013).

3. H. Puchta and F. Fauser, Gene targeting in plants: 25 yearslater. Int J Dev Biol, 57(6-8): p. 629-37, (2013).

4. Y. Luo, et al., Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammaryglands. Sci Rep, 6: p. 20657, (2016).

5. L. A. Marraffini and E. J. Sontheimer, CRISPR interference:RNA-directed adaptive immunity in bacteria and archaea. NatRev Genet, 11(3): p. 181-90, (2010).

6. Y. Ishino, et al., Nucleotide sequence of the iap gene, responsiblefor alkaline phosphatase isozyme conversion in Escherichia coli,and identification of the gene product. J Bacteriol, 169(12): p.5429-33, (1987).

7. A. Bolotin, et al., Clustered regularly interspaced shortpalindrome repeats (CRISPRs) have spacers of extrachromosomalorigin. Microbiology, 151(8): p. 2551-2561, (2005).

8. F.J. Mojica, et al., Intervening sequences of regularly spacedprokaryotic repeats derive from foreign genetic elements. J MolEvol, 60(2): p. 174-82, (2005).

9. R. Jansen, et al., Identification of genes that are associated withDNA repeats in prokaryotes. Mol Microbiol, 43(6): p. 1565-75,(2002).

10. D. H. Haft, et al., A guild of 45 CRISPR-associated (Cas) proteinfamilies and multiple CRISPR/Cas subtypes exist in prokaryoticgenomes. PLoS Comput Biol, 1(6): p. e60. 14, (2005).

11. K. S. Makarova, et al., A putative RNA-interference-basedimmune system in prokaryotes: computational analysis of thepredicted enzymatic machinery, functional analogies witheukaryotic RNAi, and hypothetical mechanisms of action. BiolDirect, 1: p. 7, (2006).

VOL. 83, NOS. 7–8 219

12. R. Barrangou, et al., CRISPR provides acquired resistanceagainst viruses in prokaryotes. Science, 315(5819): p. 1709-12,(2007).

13. S. J. Brouns, et al., Small CRISPR RNAs guide antiviral defensein prokaryotes. Science, 321(5891): p. 960-4, (2008).

14. G. Gasiunas, et al., Cas9-crRNA ribonucleoprotein complexmediates specific DNA cleavage for adaptive immunity inbacteria. Proc Natl Acad Sci U S A, 109(39): p. E2579-86,(2012).

15. R. H. Staals, et al., Structure and activity of the RNA-targetingType III-B CRISPR-Cas complex of Thermus thermophilus. MolCell, 52(1): p. 135-45, (2013).

16. E. R. Westra, et al., CRISPR immunity relies on the consecutivebinding and degradation of negatively supercoiled invader DNAby Cascade and Cas3. Mol Cell, 46(5): p. 595-605, (2012).

17. R. Barrangou and L.A. Marraffini, CRISPR-Cas systems:Prokaryotes upgrade to adaptive immunity. Mol Cell, 54(2): p.234-44, (2014).

18. A. Plagens, et al., DNA and RNA interference mechanisms byCRISPR-Cas surveillance complexes. FEMS Microbiol Rev,39(3): p. 442-63, (2015).

19. D. Rath, et al., The CRISPR-Cas immune system: biology,mechanisms and applications. Biochimie, 117: p. 119-28, (2015).

20. D. Bikard, et al., CRISPR interference can prevent naturaltransformation and virulence acquisition during in vivo bacterialinfection. Cell Host Microbe, 12(2): p. 177-86, (2012).

21. J. E. Garneau, et al., The CRISPR/Cas bacterial immune systemcleaves bacteriophage and plasmid DNA. Nature, 468(7320): p.67-71, (2010).

22. C. R. Hale, et al., RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell, 139(5): p. 945-56, (2009).

23. L. A. Marraffini and E.J. Sontheimer, CRISPR interference limitshorizontal gene transfer in staphylococci by targeting DNA.Science, 322(5909): p. 1843-5, (2008).

24. K. Chylinski, et al., Classification and evolution of type IICRISPR-Cas systems. Nucleic Acids Res, 42(10): p. 6091-105,(2014).

25. E. V. Koonin and M. Krupovic, Evolution of adaptive immunityfrom transposable elements combined with innate immunesystems. Nat Rev Genet, 16(3): p. 184-92, (2015).

26. M. Krupovic, et al., Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryoticCRISPR-Cas immunity. BMC Biol, 12: p. 36, (2014).

27. K. S. Makarova, et al., Evolution and classification of theCRISPR-Cas systems. Nat Rev Microbiol, 9(6): p. 467-77, (2011).

28. R. Barrangou, Diversity of CRISPR-Cas immune systems andmolecular machines. Genome Biol, 16: p. 247, (2015).

29. D. Bikard and L.A. Marraffini, Control of gene expression byCRISPR-Cas systems. F1000Prime Rep, 5: p. 47, (2013).

30. H. K. Ratner, T.R. Sampson and D.S. Weiss, I can see CRISPRnow, even when phage are gone: a view on alternative CRISPR-Cas functions from the prokaryotic envelope. Curr Opin InfectDis, 28(3): p. 267-74, (2015).

31. T. Wang, et al., Genetic screens in human cells using theCRISPR-Cas9 system. Science, 343(6166): p. 80-4, (2014).

32. K. S. Makarova, Y. I. Wolf and E. V. Koonin, The basic buildingblocks and evolution of CRISPR-CAS systems. Biochem SocTrans, 41(6): p. 1392-400, (2013).

33. G. Vestergaard, R. A. Garrett and S. A. Shah, CRISPR adaptiveimmune systems of Archaea. RNA Biol, 11(2): p. 156-67, (2014).

34. R. Heler, L. A. Marraffini and D. Bikard, Adapting to newthreats: the generation of memory by CRISPR-Cas immunesystems. Mol Microbiol, 93(1): p. 1-9, (2014).

35. K. A. Datsenko, et al., Molecular memory of prior infectionsactivates the CRISPR/Cas adaptive bacterial immunity system.Nat Commun, 3: p. 945, (2012).

36. R. Heler, et al., Cas9 specifies functional viral targets duringCRISPR-Cas adaptation. Nature, 519(7542): p. 199-202. 15,(2015).

37. J. K. Nunez, et al., Foreign DNA capture during CRISPR-Casadaptive immunity. Nature, 527(7579): p. 535-8, (2015).

38. J. K. Nunez, et al., Cas1-Cas2 complex formation mediatesspacer acquisition during CRISPR-Cas adaptive immunity. NatStruct Mol Biol, 21(6): p. 528-34, (2014).

39. I. Yosef, M.G. Goren and U. Qimron, Proteins and DNA elementsessential for the CRISPR adaptation process in Escherichia coli.Nucleic Acids Res, 40(12): p. 5569-76, (2012).

40. A. Plagens, et al., Characterization of the CRISPR/Cas subtypeI-A system of the hyperthermophilic crenarchaeon Thermoproteustenax. J Bacteriol, 194(10): p. 2491-500, (2012).

41. D. C. Swarts, et al., CRISPR interference directs strand specificspacer acquisition. PLoS One, 7(4): p. e35888, (2012).

42. J. van der Oost, et al., Unravelling the structural and mechanisticbasis of CRISPR-Cas systems. Nat Rev Microbiol, 12(7): p. 479-92, (2014).

43. Y. Wei, R.M. Terns and M.P. Terns, Cas9 function and hostgenome sampling in Type II-A CRISPR-Cas adaptation. GenesDev, 29(4): p. 356-61, (2015).

44. J. Carte, et al., Binding and cleavage of CRISPR RNA by Cas6.RNA, 16(11): p. 2181-8, (2010).

45. E. L. Garside, et al., Cas5d processes pre-crRNA and is amember of a larger family of CRISPR RNA endonucleases. RNA,18(11): p. 2020-8, (2012).

46. E. M. Gesner, et al., Recognition and maturation of effectorRNAs in a CRISPR interference pathway. Nat Struct Mol Biol,18(6): p. 688-92, (2011).

47. M. M. Jore, et al., Structural basis for CRISPR RNA-guidedDNA recognition by Cascade. Nat Struct Mol Biol, 18(5): p.529-36, (2011).

48. Y. Koo, et al., Conservation and variability in the structure andfunction of the Cas5d endoribonuclease in the CRISPR-mediatedmicrobial immune system. J Mol Biol, 425(20): p. 3799-810,(2013).

49. K. H. Nam, et al., Cas5d protein processes pre-crRNA andassembles into a cascade-like interference complex in subtypeI-C/Dvulg CRISPR-Cas system. Structure, 20(9): p. 1574-84,(2012).

50. J. Reeks, J. H. Naismith, and M. F. White, CRISPR interference:a structural perspective. Biochem J, 453(2): p. 155-66, (2013).

51. D. G. Sashital, M. Jinek, and J.A. Doudna, An RNA-inducedconformational change required for CRISPR RNA cleavage bythe endoribonuclease Cse3. Nat Struct Mol Biol, 18(6): p. 680-7, (2011).

52. J. Carte, et al., Cas6 is an endoribonuclease that generates guideRNAs for invader defense in prokaryotes. Genes Dev, 22(24): p.3489-96, (2008).

53. A. Hatoum-Aslan, I. Maniv and L.A. Marraffini, Matureclustered, regularly interspaced, short palindromic repeats RNA(crRNA) length is measured by a ruler mechanism anchored atthe precursor processing site. Proc Natl Acad Sci U S A, 108(52):p. 21218-22, (2011).


54. R. E. Haurwitz, et al., Sequence- and structure-specific RNAprocessing by a CRISPR endonuclease. Science, 329(5997): p.1355-8,(2010).

55. R. E. Haurwitz, S. H. Sternberg, and J. A. Doudna, Csy4 relieson an unusual catalytic dyad to position and cleave CRISPRRNA. EMBO J, 31(12): p. 2824-32, (2012).

56. O. Niewoehner, M. Jinek, and J.A. Doudna, Evolution of CRISPRRNA recognition and processing by Cas6 endonucleases. NucleicAcids Res, 42(2): p. 1341-53, (2014).

57. S. H. Sternberg, R. E. Haurwitz, and J. A. Doudna, Mechanismof substrate selection by a highly specific CRISPRendoribonuclease. RNA, 18(4): p. 661-72, (2012).

58. R. Wang, et al., Interaction of the Cas6 riboendonuclease withCRISPR RNAs: recognition and cleavage. Structure, 19(2): p.257-64, (2011).

59. B. Wiedenheft, et al., RNA-guided complex from a bacterialimmune system enhances target recognition through seedsequence interactions. Proc Natl Acad Sci U S A, 108(25): p.10092-7, (2011).

60. M. Spilman, et al., Structure of an RNA silencing complex ofthe CRISPR-Cas immune system. Mol Cell, 52(1): p. 146-52,(2013).

61. C. Peng, M. Lu, and D. Yang, CRISPR/Cas9-based tools fortargeted genome editing and replication control of HBV. VirolSin, 30(5): p. 317-25, (2015).

62. P. Samai, et al., Co-transcriptional DNA and RNA Cleavageduring Type III CRISPR-Cas Immunity. Cell, 161(5): p. 1164-74, (2015).

63. H. Deveau, et al., Phage response to CRISPR-encoded resistancein Streptococcus thermophilus. J Bacteriol, 190(4): p. 1390-400,(2008).

64. F. J. Mojica, et al., Short motif sequences determine the targetsof the prokaryotic CRISPR defence system. Microbiology, 155(3):p. 733-740, (2009).

65. J. G. Zalatan, et al., Engineering complex synthetictranscriptional programs with CRISPR RNA scaffolds. Cell,160(1-2): p. 339-50, (2015).

66. J. D. Sander, and J. K. Joung, CRISPR-Cas systems for editing,regulating and targeting genomes. Nat Biotechnol, 32(4): p. 347-55, (2014).

67. M. Jinek, et al., A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity. Science,337(6096): p. 816-21, (2012).

68. F. A. Ran, et al., Genome engineering using the CRISPR-Cas9system. Nat Protoc, 8(11): p. 2281-308, (2013).

69. S. Nielsen, Y. Yuzenkova, and N. Zenkin, Mechanism ofeukaryotic RNA polymerase III transcription termination.Science, 340(6140): p. 1577-80, (2013).

70. L. A. Gilbert, et al., Genome-Scale CRISPR-Mediated Controlof Gene Repression and Activation. Cell, 159(3): p. 647-61,(2014).

71. N. E. Sanjana, O. Shalem, and F. Zhang, Improved vectors andgenome-wide libraries for CRISPR screening. Nat Methods,11(8): p. 783-4, (2014).

72. O. Shalem, et al., Genome-scale CRISPR-Cas9 knockoutscreening in human cells. Science. 343 (6166): p. 84-7, (2014).

73. J. E. DiCarlo, et al., Genome engineering in Saccharomycescerevisiae using CRISPR-Cas systems. Nucleic Acids Res, 41(7):p. 4336-43, (2013).

74. Z. Liang, et al., Targeted mutagenesis in Zea mays using TALENsand the CRISPR/Cas system. J Genet Genomics, 41(2): p. 63-8,(2014).

75. W. Y. Hwang, et al., Efficient genome editing in zebrafish usinga CRISPR-Cas system. Nat Biotechnol, 31(3): p. 227-9, (2013).

76. Z. Yu, et al., Highly efficient genome modifications mediated byCRISPR/Cas9 in Drosophila. Genetics, 195(1): p. 289-91, (2013).

77. A. E. Friedland, et al., Heritable genome editing in C. elegansvia a CRISPR-Cas9 system. Nat Methods, 10(8): p. 741-3,(2013).

78. H. Wang, et al., One-step generation of mice carrying mutationsin multiple genes by CRISPR/Cas-mediated genome engineering.Cell, 153(4): p. 910-8, (2013).

79. H. Ebina, et al., Harnessing the CRISPR/Cas9 system to disruptlatent HIV-1 provirus. Sci Rep, 3: p. 2510, (2013).

80. R. M. Genga, N.A. Kearns and R. Maehr, Controllingtranscription in human pluripotent stem cells using CRISPR-effectors. Methods, 101: p. 36-42, (2016).

81. I. B. Hilton, et al., Epigenome editing by a CRISPR-Cas9-basedacetyltransferase activates genes from promoters and enhancers.Nat Biotechnol, 33(5) : p. 510-7, (2015).

82. P. I. Thakore, et al., Highly specific epigenome editing byCRISPR-Cas9 repressors for silencing of distal regulatoryelements. Nat Methods, 12(12), p. 1143-9, (2015).

83. F. Soldner, et al., Parkinson-associated risk variant in distalenhancer of alpha-synuclein modulates target gene expression.Nature, 533(7601), p. 95-9, (2016).

84. Y. Chen, et al., Functional disruption of the dystrophin gene inrhesus monkey using CRISPR/Cas9. Hum Mol Genet, 24(13): p.3764-74, (2015).

85. B. Chen, et al., Dynamic imaging of genomic loci in livinghuman cells by an optimized CRISPR/Cas system. Cell. 155(7):p. 1479-91, (2013).

86. W. Deng, et al., CASFISH: CRISPR/Cas9-mediated in situlabeling of genomic loci in fixed cells. Proc Natl Acad Sci U SA, 112(38), p. 11870-5, (2015).

87. K. S. Makarova, et al., Live virus-free or die: coupling ofantivirus immunity and programmed suicide or dormancy inprokaryotes. Biol Direct, 7: p. 40, (2012).

88. H. Ledford, CRISPR, the disruptor. Nature, 522(7554): p. 20-4.17, (2015).

89. L. S. Qi, et al., Repurposing CRISPR as an RNA-guided platformfor sequence-specific control of gene expression. Cell, 152(5):p. 1173-83, (2013).

90. R. Peng, G. Lin, and J. Li, Potential pitfalls of CRISPR/Cas9-mediated genome editing. FEBS J, 283(7): p. 1218-31 (2016).

91. E. Deltcheva, et al., CRISPR RNA maturation by trans-encodedsmall RNA and host factor RNase III. Nature, 471(7340): p.602-7, (2011).

92. S. H. Sternberg, et al., Conformational control of DNA targetcleavage by CRISPR-Cas9. Nature, 527(7576): p. 110-3,(2015).

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