1,10

CRISPR/Cas9 for Genome Engineering: the Next Genomic Revolution

Yaochun Zhang, Wee Song Yeo, Kar Hui Ng, Hui Kim Yap

Yaochun Zhang, Wee Song Yeo, Kar Hui Ng, Hui Kim Yap, Khoo Teck Puat-National University Children’s Medical Institute, National University Health System, Singapore
Yaochun Zhang, Wee Song Yeo, Kar Hui Ng, Hui Kim Yap, Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Correspondence to: Yaochun Zhang, Centre of Translational Medicine, MD6 Level 10 South, 14 Medical Drive, 117599, Singapore
Email: paezyc@nus.edu.sg
Telephone: +65-6601 3308
Received: April 23, 2015
Revised: October 28, 2015
Accepted: October 30, 2015
Published online: December 28, 2015

ABSTRACT

Genetically modified cells and animals represent a crucial tool for understanding gene function in development and diseases. The recently developed simple-to-design, easy-to-use and multiplexing CRISPR/Cas9 system is an efficient gene-targeting technology that has already stimulated innovative applications in biology and enabled researchers to make changes in the sequence or expression of any gene in virtually any cell type or organism of interest. When combined with large libraries of sgRNAs, CRISPR/Cas9 enables facile comprehensive forward genetic screens both in vitro and in vivo. Although challenges still remain regarding the off-target mutations, delivery methods as well as the frequency of homology-directed repair, the rapid advance in CRISPR/Cas9 technology opens the door for gene function revealing and genome and epigenome editing. It can be optimistically anticipated that, in the future, CRISPR/Cas9 technology may revolutionize gene therapy research and become a convenient and versatile tool to treat a wide variety of human diseases.

© 2015 ACT. All rights reserved.

Key words:CRISPR/Cas9; Genome Engineering; Mechanism; Application

Zhang Y, Yeo WS, Ng KH, Yap HK. CRISPR/Cas9 for Genome Engineering: the Next Genomic Revolution. Journal of Biochemistry and Molecular Biology Research 2015; 1(4): 112-117 Available from: URL: http://www.ghrnet.org/index.php/jbmbr/article/view/1615

EDITORIAL

The last two decades have witnessed a rapid progress in genetic sciences. Despite tremendous advances in high-throughput sequencing technology and the rapid generation of whole-genome sequencing data for large numbers of all types of organisms, elucidation of the underlying molecular mechanism of genes influencing individual phenotypes remains a major challenge facing the researchers. A rational way to elucidate the function of a gene or a gene mutation is to silence or overexpress it in living organisms. Conventional genetic engineering methods have generally been limited to random addition of new pieces of DNA into the host genome without any control on the insertion loci of interest and insertion copy numbers, thus not allowing the editing of specific DNA sequence in their natural context. New genome editing approaches that built on engineered, programmable and highly specific nucleases, which can induce site-specific changes in the genome of cellular organisms through a sequence-specific DNA-binding domains and a nonspecific DNA cleavage domain have emerged and are widely used in the studies of functional genomics, transgenic organism and gene therapy.

The first well studied artificial nuclease system is zinc-finger nucleases (ZFNs)[1-4]. Zinc fingers are small protein structural motifs that are typically 25-30 residues long and possess two cysteine and two histidine residues that coordinate a zinc ion. Zinc finger DNA binding proteins typically contain two or more zinc fingers with each finger interacting with 3 base pairs of DNA, typically interacting with contiguous 3-base pair recognition sites. By fusing a zinc-finger DNA-binding domain with a nonspecific cleavage domain from the type IIS restriction endonuclease FokI, ZFNs can be engineered to target desired DNA sequence and induce DNA double strand break. The nuclease domain requires dimerization to be active, and thus two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart to facilitate dimerization of the nuclease domain[1,2,5-11]. Although ZFNs are effective genome editing tools and numerous innovations to improve the utility of ZFNs have occurred, the technical challenges inherent in designing and validating engineering zinc-finger proteins for a specific DNA locus of interest have limited their use beyond by experts in the field. In 2009, the genome editing field has further expanded with the development of transcription activator-like effector nucleases (TALENs)[12,13]. TALEs are transcriptional activators that specifically bind and regulate plant genes during pathogenesis. Within the TALE structure, a repeated highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids that mediate DNA recognition. These two locations are highly variable (Repeat Variable Di-residue, RVD) and show a strong correlation with specific nucleotide recognition. Each unit of a TALE protein recognizes only a single base pair. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs, which is much easier than zinc fingers to produce and validate, enabling much more widespread applications in genome editing[14-20]. The recent emergence of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which depends on small RNA guided sequence-specific cleavage, however, has transformed the genome engineering fields by removing the need for any expertise in protein engineering[21-26].

CRISPRs were firstly discovered from the genome of Escherichia coli in 1980s and had been described as a series of direct repeats interspaced with short sequences[27]. Later studies have shown that CRISPR/Cas exists in nearly 40% genomes of sequenced bacteria and nearly 90% genomes of sequenced archaea[28]. Based on the observations that many spacer sequences within CRISPRs derive from plasmid and viral origins, CRISPRs loci can be transcribed and that cas (CRISPR-associated gene) encode proteins with putative nuclease and helicase domains, CRISPRs were predicated as an adaptive defence system that use antisense RNAs as memory signatures of past invasions[29]. However, it was not until in 2007 that infection experiments with lytic phages provided the first experimental evidence of CRISPR/Cas-mediated adaptive immunity. In those studies, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity[21]. In 2008, Brouns et al found that mature CRISPR RNAs (crRNAs) served as guides in a complex with Cas proteins to interfere with viral proliferation[22]. Functional CRISPR-Cas loci consist of a series of conserved repeated sequences interspaced by distinct non-repetitive sequences named spacers that encode the crRNA components and an operon of cas genes encoding the Cas protein components. After the first infection of viruses or phages, invading foreign DNA is processed by Cas nucleases into small DNA fragments, which are then incorporated into CRISPR locus of host genomes as the spacers. In response to virus or phage infections, the spacers are used as transcriptional templates for producing precursor crRNA (pre-crRNA) that undergoes maturation to generate individual crRNAs, each composed of a repeat portion and an invader-targeting spacer portion, which guides Cas to cleave foreign nucleic acid by Cas proteins at sites complementary to the crRNA spacer sequence. More than 40 different Cas protein families have been reported, which are primarily classified into three types (I, II, and III)[30-32]. The type II CRISPR/Cas system utilizes Cas9, a large multifunctional protein with two putative nuclease domains (HNH[29,30] and RuvC-like[29]), to cleave dsDNA, whereas Type I and Type III systems require multiple distinct effectors acting as a complex[33,34]. CRISPR/Cas9 has been demonstrated to be a simple and efficient tool for genome engineering.

In endogenous CRISPR/Cas9 system, mature crRNA is combined with trans-activating crRNA (tracrRNA), a small RNA that is trans-encoded upstream of the type II CRISPR-Cas locus and is essential for crRNA maturation by ribonuclease III and Cas9[35], to form a tracrRNA:crRNA complex that guides Cas9 to a target site. Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Importantly, Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3’ end. PAM is a short sequence motif adjacent to the crRNA-targeted sequence on the invading DNA, playing an essential role in the stages of adaptation and interference in type I and type II systems[36,37]. Different Type II systems have differing PAM requirements. The S. pyogenes system requires an NGG sequence[34], S. thermophilus Type II systems require NNAGAA for CRISPR1[23,25] and NGGNG for CRISPR3[33,38], while Neisseria meningiditis requires NNNNGATT[39]. After binding to the target site, the DNA single-strand matching crRNA and opposite strand are cleaved, respectively, by the HNH nuclease domain and RuvC-like nuclease domain of Cas9, generating a double-strand break (DSB) at the target site[33,34]. The CRISPR/Cas9 induced DSB will trigger cellular DNA repair processes, including nonhomologous end-joining (NHEJ)-mediated error-prone DNA repair and homology-directed repair (HDR)-mediated error-free DNA repair, both of which can be used to achieve a desired editing outcome. In NHEJ-mediated DNA repair, the break ends are directly ligated without the need for a homologous template, generating small insertion and deletion mutations at target sites. These mutations can help us to disrupt or abolish the function of target genes or genomic elements. If the insertion or deletion occurring within a coding exon can lead to frameshift mutations and premature stop codons, gene knockouts are achieved[40]. In contrast to NHEJ-mediated DNA repair, HDR-mediated error-free DNA repair requires a homology-containing donor DNA sequence as repair template. The repair template can either be double stranded DNA with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs). ssODNs provides an effective and simple method for making small edits in the genome, such as the introduction of single nucleotide mutations[41]. However, HDR is generally active only in dividing cells and typically occurs at lower frequencies than NHEJ[42]. For easy application in genome editing, the dual tracrRNA:crRNA was then engineered as a single guide RNA (sgRNA), which was a chimeric RNA containing all essential crRNA and tracrRNA components (Figure 1)[34]. In this two-component system, by simply changing the guide sequence of the sgRNA, it theoretically enables CRISPR/Cas9 to target any DNA sequence of interest as long as it is adjacent to a PAM. Furthermore, when Cas9 is coupled with several sgRNAs targeting on different sites, CRISPR/Cas9 is able to simultaneously induce genomic modifications at multiple genes or multiple independent sites of the same gene[25,36], accelerating the study of gene function and epistatic relationships[43-45]. For this reason, the CRISPR/Cas9 has been rapidly and widely adopted by the scientific community to target, edit, or modify the genomes of a vast array of cells and organisms.

As a robust genome editing tool, CRISPR/Cas9 enables researchers to precisely manipulate specific genomic elements not only in mammalian genomes[25,46-49] but also in the genomes of rat[50], mice[43,51], zebrafish[37,52-54], Drosophila[55-60], C. elegans[61-64], Bombyx mori[65-68], bacteria[69-71] as well as in crop plants[72-77]. Targeting with multiple sgRNAs (multiplexing) was also successfully achieved[25,48]. When combined with lentiviral sgRNA library, a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection was developed, establishing Cas9/sgRNA screening as a powerful tool for systematic genetic analysis in mammalian cells[78,79]. Recently, the genome-wide CRISPR/Cas9-mediated loss-of-function screen was further developed to systematically assay gene phenotypes in cancer evolution in vivo[80,81]. It can be anticipated that this high-throughput sequencing tool will not only facilitate the rapid identification of genes that participate in certain biological processes but will also enable large-scale screening for drug targets and other phenotypes. Another important application of CRISPR/Cas9 system is in the research of transcriptional regulation. By targeting on the transcription-related functional sites, CRISPR/Cas9 can regulate the transcription of specific genes. However, this process is irreversible due to permanent DNA modifications. To develop a CRISPR inference (CRISPRi) system for RNA-guided transcription regulation beyond permanent modification of DNA, sgRNA was co-expressed with a catalytically defective Cas9 mutant (dCas9) to form a recognition complex, which could interfere with transcriptional elongation, RNA polymerase and transcription factor binding[82-84]. Demonstrated first in E. coli, whole-genome sequencing showed that there were no detectable off-target effects[83]. CRISPRi has also been proved to repress multiple target genes simultaneously, and its effects are reversible[83,85]. After fusion with repressive or activating effector domains, dCas9 together with sgRNA, could implement precise and stable transcriptional control of target genes, including transcription repression (CRISPRi) and activation (CRISPRa) with high specificity[82,86]. Similar to those performed using RNAi, dCas9 can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, laying the foundation for future experiments involving genome-wide screening. The CRISPR/Cas9 system has also been employed for efficient correction of genetic disease[87,88]. A dominant mutation in the Crygc gene responsible for cataracts was successfully corrected in mice by co-injection of CRISPR/Cas9 with an exogenously supplied oligonucleotide into zygotes[88]. The CRISPR/Cas9 system can be further developed as an imaging tool for imaging of specific loci, by fusing dCas9 with fluorescent proteins, in live cells[89], and developed as a new therapeutic strategy against viral infections, disrupting proviruses, eliminating viral genomes and thus curing viral infections[90-93].

A few careful studies, however, duly raised concerns that CRISPR/Cas9 had tolerance to base pair mismatches between gRNA and its complementary target sequence. Cas9 can cleave the target DNA both in vitro[34] and in mammalian[25] and bacterial cells[70] when complexed with a crRNA that contains a one-base mismatch with the target sequence. More recently, three group independently showed that CRISPR/Cas9 can induce off-target mutations even the mismatch up to 5 nt[26,94,95]. Since CRISPR/Cas9 tolerates mismatches especially in the 5’ upstream region, but not in the seed region of 6-11 nt that is immediately upstream of the PAM sequence, target sites should be carefully selected so that none or very few of the seed region sequence of the designed sgRNA exist at any other location of the genome[26,94,96]. Another important factor in tolerance to mismatches is the amount of Cas9 enzyme expressed in the cells. Usually, high concentration of the enzyme increases off-site targeting, whereas lowering the concentration of Cas9 increases specificity while diminishing on-target cleavage activity[94,95]. As mentioned previously, the DNA single-strand matching crRNA and opposite strand are cleaved by the HNH nuclease domain and RuvC-like nuclease domain of Cas9 respectively[33,34], mutating either domain in Cas9 generates a variant protein with single-stranded DNA cleavage (nickase) activity. With the principle that two adjacent off-target binding events and subsequent cleavage are less likely to occur than a single off-target cleavage, Cas9 nickase with paired gRNAs properly positioned on the target DNA exhibits low off-target mutagenesis compared to wild-type Cas9[97-102]. Also there are studies use an sgRNA-guided dCas9 fused to the FokI nuclease where two fused dCas9-FokI bind target sites at a defined distance apart, inducing DNA double strand break after dimerization of the two monomers[103,104]. Additionally, following the reasons that the 5′-end nucleotides of the sgRNAs are not necessary for their full activity, however, they may compensate for mismatches at other positions along the guide RNA-target DNA interface, leading to off-target mutations, shorter sgRNAs truncated by two or three nucleotides at the distal end relative to the PAM can be used in the double nicking strategy to further reduce off-target activity[105].

conclusion

In conclusion, the RNA-guided, two component CRISPR/Cas9 system offers several advantages over the previous protein guided counterparts. The simple-to-design, easy-to-use and multiplexing technology has already stimulated innovative applications in biology and enabled researchers to make changes in the sequence or expression of any gene in virtually any cell type or organism of interest. When combined with large libraries of sgRNAs, CRISPR/Cas9 enables facile comprehensive forward genetic screens both in vitro and in vivo. Furthermore, this gene editing tool has shown therapeutic potentials for genic diseases, infectious diseases and cancer. Despite the great potential of CRISPR/Cas9 in genome editing, challenges still remain regarding the off-target mutations, delivery methods as well as the frequency of homology-directed repair. However, with the rapid advance in CRISPR/Cas9 technology, it opens the door for gene function revealing and genome and epigenome editing. It can be optimistically anticipated that, in the future, CRISPR/Cas9 technology may revolutionize gene therapy research and become a convenient and versatile tool to treat a wide variety of human diseases.

CONFLICT OF INTERESTS

The authors have no conflicts of interest to declare.

REFERENCES

1.Alwin S, Gere MB, Guhl E, et al. Custom zinc-finger nucleases for use in human cells. Mol Ther 2005;12:610-7.

2.Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic acids research 2005;33:5978-90.

3.Mani M, Kandavelou K, Dy FJ, Durai S, Chandrasegaran S. Design, engineering, and characterization of zinc finger nucleases. Biochemical and biophysical research communications 2005;335:447-57.

4.Porteus MH, Carroll D. Gene targeting using zinc finger nucleases. Nature biotechnology 2005;23:967-73.

5.Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005;435:646-51.

6.Carroll D, Morton JJ, Beumer KJ, Segal DJ. Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 2006;1:1329-41.

7.Porteus MH. Mammalian gene targeting with designed zinc finger nucleases. Mol Ther 2006;13:438-46.

8.Lombardo A, Genovese P, Beausejour CM, et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nature biotechnology 2007;25:1298-306.

9.Cathomen T, Segal DJ, Brondani V, Muller-Lerch F. Generation and functional analysis of zinc finger nucleases. Methods Mol Biol 2008;434:277-90.

10.Weinthal D, Tovkach A, Zeevi V, Tzfira T. Genome editing in plant cells by zinc finger nucleases. Trends Plant Sci 2010;15:308-21.

11.Zeevi V, Tovkach A, Tzfira T. Artificial zinc finger nucleases for DNA cloning. Methods Mol Biol 2010;649:209-25.

12.Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009;326:1509-12.

13.Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science 2009;326:1501.

14.Zhang M, Wang F, Li S, Wang Y, Bai Y, Xu X. TALE: a tale of genome editing. Progress in biophysics and molecular biology 2014;114:25-32.

15.Cermak T, Doyle EL, Christian M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research 2011;39:e82.

16.Bedell VM, Wang Y, Campbell JM, et al. In vivo genome editing using a high-efficiency TALEN system. Nature 2012;491:114-8.

17.Ding Q, Lee YK, Schaefer EA, et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 2013;12:238-51.

18.Li T, Yang B. TAL effector nuclease (TALEN) engineering. Methods Mol Biol 2013;978:63-72.

19.Zu Y, Tong X, Wang Z, et al. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nature methods 2013;10:329-31.

20.Wang H, Hu YC, Markoulaki S, et al. TALEN-mediated editing of the mouse Y chromosome. Nature biotechnology 2013;31:530-2.

21.Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315:1709-12.

22.Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008;321:960-4.

23.Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010;468:67-71.

24.Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science 2010;327:167-70.

25.Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819-23.

26.Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology 2013;31:822-6.

27.Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 1987;169:5429-33.

28.Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC bioinformatics 2007;8:172.

29.Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology direct 2006;1:7.

30.Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS computational biology 2005;1:e60.

31.Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biology direct 2011;6:38.

32.Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems. Nature reviews Microbiology 2011;9:467-77.

33.Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 2012;109:E2579-86.

34.Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-21.

35.Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471:602-7.

36.Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell research 2013;23:1163-71.

37.Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Sciences of the United States of America 2013;110:13904-9.

38.Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research 2011;39:9275-82.

39.Zhang Y, Heidrich N, Ampattu BJ, et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Molecular cell 2013;50:488-503.

40.Burma S, Chen BP, Chen DJ. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst) 2006;5:1042-8.

41.Chen F, Pruett-Miller SM, Huang Y, et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature methods 2011;8:753-5.

42.Saleh-Gohari N, Helleday T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic acids research 2004;32:3683-8.

43.Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013;153:910-8.

44.Fujii W, Onuma A, Sugiura K, Naito K. One-step generation of phenotype-expressing triple-knockout mice with heritable mutated alleles by the CRISPR/Cas9 system. J Reprod Dev 2014;60:324-7.

45.Zhou J, Shen B, Zhang W, et al. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol 2014;46:49-55.

46.Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. Elife 2013;2:e00471.

47.Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature biotechnology 2013;31:230-2.

48.Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science 2013;339:823-6.

49.Hou Z, Zhang Y, Propson NE, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proceedings of the National Academy of Sciences of the United States of America 2013;110:15644-9.

50.Chapman KM, Medrano GA, Jaichander P, et al. Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells. Cell Rep 2015;10:1828-35.

51.Shen B, Zhang J, Wu H, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell research 2013;23:720-3.

52.Hruscha A, Schmid B. Generation of zebrafish models by CRISPR /Cas9 genome editing. Methods Mol Biol 2015;1254:341-50.

53.Hisano Y, Sakuma T, Nakade S, et al. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Scientific reports 2015;5:8841.

54.Ablain J, Durand EM, Yang S, Zhou Y, Zon LI. A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish. Dev Cell 2015;32:756-64.

55.Gratz SJ, Cummings AM, Nguyen JN, et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 2013;194:1029-35.

56.Bassett AR, Tibbit C, Ponting CP, Liu JL. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 2013;4:220-8.

57.Xue Z, Wu M, Wen K, et al. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila. G3 2014;4:2167-73.

58.Gokcezade J, Sienski G, Duchek P. Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila. G3 2014;4:2279-82.

59.Zhang X, Koolhaas WH, Schnorrer F. A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila. G3 2014;4:2409-18.

60.Ren X, Yang Z, Xu J, et al. Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell Rep 2014;9:1151-62.

61.Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature methods 2013;10:741-3.

62.Zhao P, Zhang Z, Ke H, Yue Y, Xue D. Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system. Cell research 2014;24:247-50.

63.Liu P, Long L, Xiong K, et al. Heritable/conditional genome editing in C. elegans using a CRISPR-Cas9 feeding system. Cell research 2014;24:886-9.

64.Shen Z, Zhang X, Chai Y, et al. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev Cell 2014;30:625-36.

65.Wang Y, Li Z, Xu J, et al. The CRISPR/Cas system mediates efficient genome engineering in Bombyx mori. Cell research 2013;23:1414-6.

66.Ma S, Chang J, Wang X, et al. CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori. Scientific reports 2014;4:4489.

67.Liu Y, Ma S, Wang X, et al. Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9. Insect Biochem Mol Biol 2014;49:35-42.

68.Wei W, Xin H, Roy B, Dai J, Miao Y, Gao G. Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori. PloS one 2014;9:e101210.

69.DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research 2013;41:4336-43.

70.Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 2013;31:233-9.

71.Dy RL, Pitman AR, Fineran PC. Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria. Mobile genetic elements 2013;3:e26831.

72.Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 2013;9:39.

73.Kumar V, Jain M. The CRISPR-Cas system for plant genome editing: advances and opportunities. J Exp Bot 2015;66:47-57.

74.Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 2015;32:76-84.

75.Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 2015;33:41-52.

76.Ali Z, Abul-Faraj A, Li L, et al. Efficient Virus-Mediated Genome Editing in Plants using the CRISPR/Cas9 System. Molecular plant 2015.

77.Li JF, Zhang D, Sheen J. Targeted Plant Genome Editing via the CRISPR/Cas9 Technology. Methods Mol Biol 2015;1284:239-55.

78.Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science 2014;343:80-4.

79.Zhou Y, Zhu S, Cai C, et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 2014;509:487-91.

80.Burgess DJ. Cancer genetics: CRISPR screens go in vivo. Nat Rev Genet 2015;16:194-5.

81.Chen S, Sanjana NE, Zheng K, et al. Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis. Cell 2015;160:1246-60.

82.Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154:442-51.

83.Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152:1173-83.

84.Malina A, Mills JR, Cencic R, et al. Repurposing CRISPR/Cas9 for in situ functional assays. Genes Dev 2013;27:2602-14.

85.Choudhary E, Thakur P, Pareek M, Agarwal N. Gene silencing by CRISPR interference in mycobacteria. Nat Commun 2015;6:6267.

86.Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 2014;159:647-61.

87.Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature biotechnology 2014;32:551-3.

88.Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 2013;13:659-62.

89.Chen B, Gilbert LA, Cimini BA, et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 2013;155:1479-91.

90.Zhu W, Lei R, Le Duff Y, et al. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology 2015;12:22.

91.Liao HK, Gu Y, Diaz A, et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat Commun 2015;6:6413.

92.Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Scientific reports 2013;3:2510.

93.Hu W, Kaminski R, Yang F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proceedings of the National Academy of Sciences of the United States of America 2014;111:11461-6.

94.Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology 2013;31:827-32.

95.Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature biotechnology 2013;31:839-43.

96.Xiao A, Cheng Z, Kong L, et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 2014.

97.Duda K, Lonowski LA, Kofoed-Nielsen M, et al. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic acids research 2014;42:e84.

98.Cho SW, Kim S, Kim Y, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 2014;24:132-41.

99.Lee AY, Lloyd KC. Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice. FEBS Open Bio 2014;4:637-42.

100.Ren X, Yang Z, Mao D, et al. Performance of the Cas9 nickase system in Drosophila melanogaster. G3 2014;4:1955-62.

101.Rong Z, Zhu S, Xu Y, Fu X. Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template. Protein Cell 2014;5:258-60.

102.Shen B, Zhang W, Zhang J, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods 2014;11:399-402.

103.Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature biotechnology 2014;32:577-82.

104.Tsai SQ, Wyvekens N, Khayter C, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature biotechnology 2014;32:569-76.

105.Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature biotechnology 2014;32:279-84.

Peer reviewer:Janney Sun, Executive Editor-In-Chief, Unit A1, 7/F, Cheuk Nang Plaza, 250 Hennessy Road, Wanchai, Hong Kong.

Refbacks

  • There are currently no refbacks.