CRISPR/Cas9 & Targeted Genome Editing: New Era in Molecular Biology (2024)

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goalfor biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcuspyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, includinghom*ologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensiveand high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene functionand unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] andtranscription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestrandedbreaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use,particularly for large scale, high-throughput studies.

What is CRISPR/Cas9?

The functions of CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats) andCRISPR-associated (Cas) genes are essential inadaptive immunity in select bacteria and archaea,enabling the organisms to respond to andeliminate invading genetic material. These repeatswere initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmeduntil 2007 by Barrangou and colleagues, whodemonstrated that S. thermophilus can acquire resistanceagainst a bacteriophage by integratinga genome fragment of an infectious virus into itsCRISPR locus (10).

Three types of CRISPR mechanisms have beenidentified, of which type II is the most studied. Inthis case, invading DNA from viruses or plasmidsis cut into small fragments and incorporated intoa CRISPR locus amidst a series of short repeats(around 20 bps). The loci are transcribed, andtranscripts are then processed to generate smallRNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that targetinvading DNA based on sequence complementarity(Figure 1) (11).

One Cas protein, Cas9 (also known as Csn1),has been shown, through knockdown and rescueexperiments to be a key player in certain CRISPRmechanisms (specifically type II CRISPR systems).The type II CRISPR mechanism is unique comparedto other CRISPR systems, as only one Cas protein(Cas9) is required for gene silencing (12). In typeII systems, Cas9 participates in the processing ofcrRNAs (12), and is responsible for the destruction ofthe target DNA (11). Cas9’s function in both of thesesteps relies on the presence of two nuclease domains,a RuvC-like nuclease domain located at the aminoterminus and a HNH-like nuclease domain that residesin the mid-region of the protein (13).

To achieve site-specific DNA recognition andcleavage, Cas9 must be complexed with both acrRNA and a separate trans-activating crRNA(tracrRNA or trRNA), that is partially complementaryto the crRNA (11). The tracrRNA is requiredfor crRNA maturation from a primary transcriptencoding multiple pre-crRNAs. This occurs in thepresence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNHand RuvC-like nuclease domains cut both DNAstrands, generating double-stranded breaks (DSBs)at sites defined by a 20-nucleotide target sequencewithin an associated crRNA transcript (11, 14).The HNH domain cleaves the complementarystrand, while the RuvC domain cleaves the noncomplementarystrand.

The double-stranded endonuclease activity of Cas9also requires that a short conserved sequence, (2–5nts) known as protospacer-associated motif (PAM),follows immediately 3´- of the crRNA complementarysequence (15). In fact, even fully complementarysequences are ignored by Cas9-RNA in theabsence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool inMolecular Biology

The simplicity of the type II CRISPR nuclease,with only three required components (Cas9 alongwith the crRNA and trRNA) makes this systemamenable to adaptation for genome editing. Thispotential was realized in 2012 by the Doudnaand Charpentier labs (11). Based on the type IICRISPR system described previously, the authorsdeveloped a simplified two-component systemby combining trRNA and crRNA into a singlesynthetic single guide RNA (sgRNA). sgRNAprogrammedCas9 was shown to be as effectiveas Cas9 programmed with separate trRNA andcrRNA in guiding targeted gene alterations(Figure 2A).

To date, three different variants of the Cas9nuclease have been adopted in genome-editingprotocols. The first is wild-type Cas9, whichcan site-specifically cleave double-strandedDNA, resulting in the activation of the doublestrandbreak (DSB) repair machinery. DSBs canbe repaired by the cellular Non-hom*ologousEnd Joining (NHEJ) pathway (17), resultingin insertions and/or deletions (indels) whichdisrupt the targeted locus. Alternatively, if a donor template with hom*ology to the targetedlocus is supplied, the DSB may be repaired bythe hom*ology-directed repair (HDR) pathwayallowing for precise replacement mutations to bemade (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 systema step further towards increased precision bydeveloping a mutant form, known as Cas9D10A,with only nickase activity. This means it cleavesonly one DNA strand, and does not activateNHEJ. Instead, when provided with a hom*ologousrepair template, DNA repairs are conducted viathe high-fidelity HDR pathway only, resulting inreduced indel mutations (1, 11, 19). Cas9D10A iseven more appealing in terms of target specificitywhen loci are targeted by paired Cas9 complexesdesigned to generate adjacent DNA nicks (20) (seefurther details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9(dCas9, Figure 2C) (21). Mutations H840A in theHNH domain and D10A in the RuvC domaininactivate cleavage activity, but do not preventDNA binding (11, 22). Therefore, this variantcan be used to sequence-specifically target anyregion of the genome without cleavage. Instead, by fusing with various effector domains, dCas9can be used either as a gene silencing or activationtool (21, 23–26). Furthermore, it can be usedas a visualization tool. For instance, Chen andcolleagues used dCas9 fused to Enhanced GreenFluorescent Protein (EGFP) to visualize repetitiveDNA sequences with a single sgRNA or nonrepetitiveloci using multiple sgRNAs (27).

Targeting Efficiency and Off-targetMutations

Targeting efficiency, or the percentage of desiredmutation achieved, is one of the most importantparameters by which to assess a genome-editingtool. The targeting efficiency of Cas9 comparesfavorably with more established methods, suchas TALENs or ZFNs (8). For example, in humancells, custom-designed ZFNs and TALENs couldonly achieve efficiencies ranging from 1% to50% (29–31). In contrast, the Cas9 system hasbeen reported to have efficiencies up to >70%in zebrafish (32) and plants (33), and rangingfrom 2–5% in induced pluripotent stem cells (34).In addition, Zhou and colleagues were able toimprove genome targeting up to 78% in one-cellmouse embryos, and achieved effective germlinetransmission through the use of dual sgRNAs tosimultaneously target an individual gene (35).

A widely used method to identify mutations is theT7 Endonuclease I mutation detection assay (36,37) (Figure 3). This assay detects heteroduplexDNA that results from the annealing of a DNAstrand, including desired mutations, with a wildtypeDNA strand (37).

Another important parameter is the incidence ofoff-target mutations. Such mutations are likely toappear in sites that have differences of only a fewnucleotides compared to the original sequence,as long as they are adjacent to a PAM sequence.This occurs as Cas9 can tolerate up to 5 basemismatches within the protospacer region (36) ora single base difference in the PAM sequence (38).Off-target mutations are generally more difficult todetect, requiring whole-genome sequencing to rulethem out completely.

Recent improvements to the CRISPR system forreducing off-target mutations have been madethrough the use of truncated gRNA (truncatedwithin the crRNA-derived sequence) or by addingtwo extra guanine (G) nucleotides to the 5´ end(28, 37). Another way researchers have attemptedto minimize off-target effects is with the use of“paired nickases” (20). This strategy uses D10ACas9 and two sgRNAs complementary to theadjacent area on opposite strands of the target site(Figure 2B). While this induces DSBs inthe target DNA, it is expected to create only single nicks in off-target locations and, therefore, resultin minimal off-target mutations.

By leveraging computation to reduce off-targetmutations, several groups have developed webbasedtools to facilitate the identification ofpotential CRISPR target sites and assess theirpotential for off-target cleavage. Examplesinclude the CRISPR Design Tool (38) and theZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editingand Genome Targeting Tool

Following its initial demonstration in 2012(9), the CRISPR/Cas9 system has been widelyadopted. This has already been successfully usedto target important genes in many cell lines andorganisms, including human (34), bacteria (41),zebrafish (32), C. elegans (42), plants (34), Xenopustropicalis (43), yeast (44), Drosophila (45), monkeys(46), rabbits (47), pigs (42), rats (48) and mice(49). Several groups have now taken advantage ofthis method to introduce single point mutations(deletions or insertions) in a particular targetgene, via a single gRNA (14, 21, 29). Using apair of gRNA-directed Cas9 nucleases instead,it is also possible to induce large deletions orgenomic rearrangements, such as inversionsor translocations (50). A recent excitingdevelopment is the use of the dCas9 versionof the CRISPR/Cas9 system to target proteindomains for transcriptional regulation (26, 51,52), epigenetic modification (25), and microscopicvisualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesignof the crRNA to change target specificity.This contrasts with other genome editing tools,including zinc finger and TALENs, where redesignof the protein-DNA interface is required.Furthermore, CRISPR/Cas9 enables rapidgenome-wide interrogation of gene functionby generating large gRNA libraries (51, 53) forgenomic screening.

The future of CRISPR/Cas9

The rapid progress in developing Cas9 into a setof tools for cell and molecular biology researchhas been remarkable, likely due to the simplicity,high efficiency and versatility of the system. Of thedesigner nuclease systems currently available forprecision genome engineering, the CRISPR/Cassystem is by far the most user friendly. It is nowalso clear that Cas9’s potential reaches beyondDNA cleavage, and its usefulness for genomelocus-specific recruitment of proteins will likelyonly be limited by our imagination.

View NEB's Genome Editing Products

References

1. Cong L., et al. (2013) Science, 339, 819–823.
2. Capecchi, M.R. (2005) Nat. Rev. Genet. 6, 507–512.
3. Fire, A., et al. (1998) Nature, 391, 806–811.
4. Elbashir, S.Mm, et al. (2002) Methods, 26, 199–213.
5. Martinez, J., et al. (2003) Nucleic Acids Res. Suppl. 333.
6. Alic, N, et al. (2012) PLoS One, 7, e45367.
7. Miller, J., et al. (2005) Mol. Ther. 11, S35–S35.
8. Mussolino, C., et al. (2011) Nucleic Acids Res. 39, 9283–9293.
9. Ishino, Y., et al. (1987) J. Bacteriol. 169, 5429–5433.
10. Barrangou, R., et al. (2007). Science, 315, 1709–1712.
11. Jinek, M., et al. (2012) Science, 337, 816–821.
12. Deltcheva, E., et al. (2011) Nature, 471, 602–607.
13. Sapranauskas, R., et al. (2011) Nucleic Acids Res. 39, 9275–9282.
14. Nishimasu, H., et al. (2014) Cell, doi:10.1016/j.cell.2014.02.001
15. Swarts, D.C., et al. (2012) PLoS One, 7:e35888.
16. Sternberg, S.H., et al. (2014) Nature, doi:10.1038/nature13011.
17. Overballe-Petersen, S., et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110,19860–19865.
18. Gong, C., et al. (2005) Nat. Struct. Mol. Biol. 12, 304–312.
19. Davis, L., Maizels, N. (2014) Proc. Natl. Acad. Sci. U S A, 111, E924–932.
20. Ran, F.A., et al. (2013) Cell, 154, 1380–1389.
21. Qi, L.S., et al. (2013) Cell, 152, 1173–1183.
22. Gasiunas, G., et al. (2012) Proc. Natl. Acad. Sci. U S A, 109, E2579–2586.
23. Maede, M.L., et al. (2013) Nat. Methods, 10, 977–979.
24. Gilbert, L.A., et al. (2013) Cell, 154, 442–451.
25. Hu, J., et al. (2014) Nucleic Acids Res. doi:10.1093/nar/gku109.
26. Perez-Pinera, P., et al. (2013) Nat. Methods, 10, 239–242.
27. Chen, B., et al. (2013) Cell, 155, 1479–1491.
28. Seung, W., et al. (2014) Genome Res. 24, 132–141.
29. Miller, J.C., et al. (2011). Nat. Biotechnol. 29, 143–148.
30. Mussolino, C., et al. (2011). Nucleic Acids Res. 39, 9283–9293.
31. Maeder, M.L., et al. (2008) Mol. Cell, 31, 294–301.
32. Hwang, W.Y., et al. (2013) PLoS One, 8:e68708.
33. Feng, Z., et al. (2013) Cell Res. 23, 1229–1232.
34. Mali, P., et al. (2013) Science, 339, 823–826.
35. Zhou, J., et al. (2014) FEBS J. doi:10.1111/febs.12735.
36. Fu, Y., et al. (2013) Nat. Biotechnol. 31, 822–826.
37. Fu, Y., et al. (2014) Nat Biotechnol. doi: 10.1038/nbt.2808.
38. Hsu, P.D., et al. (2013) Nat. Biotechnol. 31, 827–832.
39. Sander, J.D., et al. (2007) Nucleic Acids Res. 35, W599-605.
40. Sander, J.D., et al (2010) Nucleic Acids Res. 38, W462–468.
41. Pyne M.E., et al. (2015)Appl Environ Microbiol81:5103–5114.
42. Oh J.H., et al. (2014)Nucleic Acids Res42:e131.
43. Jiang W., et al. (2013)Nat Biotechnol31:233–239.
44. Hai, T., et al. (2014) Cell Res. doi: 10.1038/cr.2014.11.
45. Guo, X., et al. (2014) Development, 141, 707–714.
46. DiCarlo, J.E., et al. (2013) Nucleic Acids Res. 41, 4336–4343.
47. Gratz, S.J., et al. (2014) Genetics, doi:10.1534/genetics.113.160713.
48. Niu, Y., et al. (2014) Cell, 156, 836–843.
49. Yang, D., et al. (2014) J. Mol. Cell Biol. 6, 97-99.
50. Ma, Y., et al. (2014) Cell Res. 24, 122–125.
51. Mashiko, D., et al. (2014) Dev. Growth Differ. 56, 122–129.
52. Gratz, S.J., et al. (2013) Fly, 249.
53. Mali, P., et al. (2013) Nat. Biotechnol. 31, 833–838.
54. Cheng, A.W., et al. (2013) Cell Res. 23, 1163–1171.
55. Koike-Yusa, H., et al. (2013) Nat. Biotechnol. doi: 10.1038/nbt.2800.
56. Sander, J.D., and Joung, J.K. (2014) Nat Biotechnol. doi:10.1038/nbt.2842.

From NEB expressions Issue I, 2014
Article by Alex Reis, Ph.D., Bitesize Bio
Breton Hornblower, Ph.D., Brett Robb, Ph.D.and George Tzertzinis, Ph.D., New England
Biolabs, Inc.