Human Molecular Genetics, 2014, Vol.

23, Review Issue 1

doi:10.1093/hmg/ddu125
Advance Access published on March 20, 2014

R40R46

CRISPR/Cas9 for genome editing: progress,

implications and challenges
Feng Zhang, Yan Wen and Xiong Guo
Key Laboratory of Environment and Gene Related Diseases of Ministry Education, Faculty of Public Health,
College of Medicine, Xian Jiaotong University, Xian, PR China
Received January 27, 2014; Revised January 27, 2014; Accepted March 17, 2014

INTRODUCTION
Benefiting from the rapid development of high-throughput sequencing technology and bioinformatics, researchers make
great progress on gene mapping in a short time. Currently, a
major challenge faced by researchers is how to reveal the molecular mechanism of genes influencing individual phenotypes.
A good way to elucidate the function of a gene is to shut it down
or overexpress it in living organisms, which is previously complicated and time consuming (1 4). A new approach named
genome editing emerged and widely used in the studies of functional genomics, transgenic organisms and gene therapy during
the past decades. Genome editing is built on engineered, programmable and highly specific nucleases, which can induce sitespecific changes in the genomes of cellular organisms through a
sequence-specific DNA-binding domain and a nonspecific DNA
cleavage domain. Subsequent cellular DNA repair process generates desired insertions, deletions or substitutions at the loci of
interest.
Multiple artificial nuclease systems have been developed for
genome editing. Zinc-finger nucleases (ZFNs) are one of
widely applied engineered nucleases (5 11). ZFNs contain a
common Cys2-His2 DNA-binding domain and a DNA cleavage
domain of the FokI restriction endonuclease (8). Another
popular genome editing platform is transcription activator-like
effector nucleases (TALENs) (12 19), which are derived from

a natural protein of plant pathogenic bacteria Xanthomonas.

The DNA-binding domain of TALENs is composed of 33 35
conserved amino acid repeated motifs, each of which recognizes
a specific nucleotide. Through shuffling repeated amino acid recognition motifs, TALENs can be programmed to target-specific
DNA sequence. Recently, clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides an alternative to ZFNs and TALENs for
genome editing (20). Distinct from the protein-guided DNA
cleavage of ZFNs and TALENs, CRISPR/Cas9 depends on
small RNA for sequence-specific cleavage (21). Because only
programmable RNA is required to generate sequence specificity,
CRISPR/Cas9 is easily applicable and develops very fast over
the past year. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome
editing and clinical therapeutic potential of CRISPR/Cas9 in
future.

CRISPR/CAS9-MEDIATED GENOME
MODIFICATION
In bacteria and archaea, CRISPR/Cas was discovered as an
acquired immune system against viruses and phages through
CRISPR RNA (crRNA)-based DNA recognition and Cas
nucleases-mediated DNA cleavage (21,22). CRISPR/Cas is

To whom correspondence should be addressed. Tel: +86 02982655091; Fax: +86 02982655332; Email: guox@mail.xjtu.edu.cn

# The Author 2014. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system
provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific
genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9
comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA),
which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA
repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/
Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future.

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

observed in nearly 40% genomes of sequenced bacteria and

nearly 90% genomes of sequenced archaea (23). CRISPR
locus consists of a series of conserved repeated sequences interspaced by distinct nonrepetitive sequences named spacers
(Fig. 1A). In CRISPR/Cas system, invading foreign DNA is processed by Cas nuclease into small DNA fragments, which are
then incorporated into CRISPR locus of host genomes as the
spacers. In response to viruses and phage infections, the spacers
are used as transcriptional templates for producing crRNA,
which guides Cas to cleave target DNA sequences of invading
viruses and phages (Fig. 1B). More than 40 different Cas protein
families have been reported (24), playing important roles in
crRNA biogenesis, spacers incorporation and invading DNA
cleavage. Based on the sequences and structures of Cas protein,
CRISPR/Cas system is primarily classified into three types, I, II
and III (25). The type II CRISPR/Cas system only needs a single
Cas protein Cas9, which contains a HNH nuclease domain and

a RuvC-like nuclease domain (21). CRISPR/Cas9 has been

demonstrated to be a simple and efficient tool for genome
editing.
CRISPR/Cas9-mediated genome editing depends on the generation of double-strand break (DSB) and subsequent cellular
DNA repair process. In endogenous CRISPR/Cas9 system,
mature crRNA is combined with transactivating crRNA (tracrRNA) to form a tracrRNA:crRNA complex that guides Cas9 to
a target site. TracrRNA is partially complementary to crRNA
and contributes to crRNA maturation. At the target site, CRISPR/
Cas9-mediated sequence-specific cleavage requires a DNA sequence protospacer matching crRNA and a short protospacer
adjacent motif (PAM). After binding to the target site, the
DNA single-strand matching crRNA and opposite strand are
cleaved, respectively, by the HNH nuclease domain and RuvClike nuclease domain of Cas9, generating a DSB at the target site
(Fig. 2). For easy application in genome editing, researchers
designed a delicate guide RNA (gRNA), which was a chimeric
RNA containing all essential crRNA and tracrRNA components
(21). Multiple CRISPR/Cas9 variants have been developed, recognizing 20 or 24 nt sequences matching engineered gRNA and
2 4 nt PAM sequences at target sites. Therefore, CRISPR/Cas9
can theoretically target a specific DNA sequence with 22 29 nt,
which is unique in most genomes. However, recent studies
observed that CRISPR/Cas9 had high tolerance to base pair mismatches between gRNA and its complementary target sequence,
which was sensitive to the numbers, positions and distribution of
mismatches (21,26 29). For instance, the CRISPR/Cas9 of
Streptococcus pyogenes appeared to tolerate up to six base
pair mismatches at target sites (21).
The DSB generated by CRISPR/Cas9 will trigger cellular
DNA repair processes, including nonhomologous end-joining
(NHEJ)-mediated error-prone DNA repair and homologydirected repair (HDR)-mediated error-free DNA repair. NHEJmediated DNA repair can rapidly ligate the DSB but generate
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. For instance, Gratz et al. generated frame-shifting indels at the yellow locus of Drosophila
genome through CRISPR/Cas9-induced DNA cleavage following by NHEJ-mediated DNA repair (30). DSB can also initiate
HDR-mediated DNA repair, which is more complicated than
NHEJ-mediated DNA repair. HDR-mediated error-free DNA
repair requires a homology-containing donor DNA sequence
as repair template. Through co-injection of Cas9, two gRNA targeting, respectively, the 5 and 3 sequences of the yellow locus,
and a single-strand oligodeoxynucleotide template, Gratz et al.
successfully replaced the yellow locus with a 50 nt attP recombination site in Drosophila genome (30).
Comparing with ZFNs and TALENs, there are several advantages for CRISPR/Cas9. ZFNs and TALENs are built on proteinguided DNA cleavage, which needs complex and time-consuming
protein engineering, selection and validation. In contrast, CRISPR/
Cas9 only needs a short programmable gRNA for DNA targeting, which is relative cheap and easy to design and produce.
Through using Cas9 and several gRNA with different target
sites, CRISPR/Cas9 is able to simultaneously induce genomic
modifications at multiple independent sites (26). This technology can accelerate the generation of transgenic animals with
multiple gene mutations (31,32), and disrupt multiple genes or

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

Figure 1 Overview of CRISPR/Cas bacterial immune system. (A) A typical

structure of CRISPR locus; (B) illustration of new spacer acquisition and invading DNA cleavage.

R41

R42

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

a whole gene family to investigate gene function and epistatic

relationships.

APPLICATIONS
Genome editing
CRISPR/Cas9 provides a robust and multiplexable genome
editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the function elucidation of
target genes in biology and diseases. Through co-delivery of
plasmids expressing Cas9 and crRNA, CRISPR/Cas9 has been
used to induce specific genomic modifications in human cells
(26,33 36). Through integrating multiple distinct gRNA with
Cas9 in a CRISPR array, CRISPR/Cas9 can simultaneously
induce multiple mutations in mammalian genomes (26). In addition to mammalian genomes, CRISPR/Cas9 also demonstrates
its potentiality in the genome editing of zebrafish (37 41), mice
(31,42), drosophila (30,43,44), caenorhabditis elegans (45),
Bombyx mori (46) and bacteria (47,48). For instance, Bassett
et al. provided an improved RNA injection-based CRISPR/

Cas9 system, which was highly efficient for creating desired

mutagenesis in Drosophila genome (43). Through directly
injecting Cas9 mRNA and gRNA into embryo, they successfully
induced mutagenesis at target sites in up to 88% of injected flies.
The generated mutations were stably transmitted to 33% of total
offspring through the germline (43).
CRISPR/Cas9 is also used to induce desired genomic alterations in plants for generating specific traits, such as valuable phenotypes or disease resistance (4957). To validate the application
of CRISPR/Cas9 in plants, Jiang et al. transferred green fluorescence protein gene into Arabidopsis and tobacco genomes, and
bacterial blight susceptibility genes into rice genome (57). Miao
et al. illustrated the robustness and efficiency of CRISPR/Cas9
in the genome editing of rice (56). Through modification of crop
genomes, CRISPR/Cas9 can be used to improve crop quality as
a new breeding technique in future.
Transcription regulation
Gene transcription regulation in living organisms is very useful
for gene function and transcriptional network studies. Through

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

Figure 2 Schematic of CRISPR/Cas9-mediated DNA cleavage. Mature crRNA guides Cas9 to the target site of invading phage DNA. The DNA single-strand matching crRNA and opposite strand are cut, respectively, by the HNH nuclease domain and RuvC-like nuclease domain of Cas9, generating a DSB at the target site. The
specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequence matching crRNA and a 3 nt PAM locating at downstream of the target sequence.

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

Gene therapy
Precisely genome editing has the potential to permanently cure
diseases through disrupting endogenous disease-causing
genes, correcting disease-causing mutations or inserting new
protective genes (61 66). Using ZFNs-induced HDR, Urnov
et al. corrected disease-causing gene mutation in human cell
for the first time (61). Subsequently, ZFNs were used to
correct the gene mutations causing sickle-cell disease (63) and
hemophilia B (62). Through disabling virulence genes or inserting protective genes, ZFNs have been used to induce resistance
to virus infection in human cells (67 69) and enhance the efficiency of immunotherapies (70,71). As the newest engineered
nucleases, CRISPR/Cas9 provides a novel highly efficient
genome editing tool for gene therapy studies. For instance,
Ebina et al. disrupted the long-terminal repeat promoter of
HIV-1 genome using CRISPR/Cas9, which significantly
decreased HIV-1 expression in infected human cells (72). The
integrated proviral viral genes in host cell genomes can also be
removed by CRISPR/Cas9 (72).
With the rapid development of induced pluripotent stem (iPS)
cells technology, engineered nucleases are applied to genome
manipulation of iPS cells (73,74). The unlimited self-renewing
and multipotential differentiation capacity of iPS cells make
them very useful in disease modeling and gene therapy. Using
CRISPR/Cas9, Horri et al. created an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) causing by DNMT3B gene mutation (75).
In this study, iPS cells were transfected with plasmids expressing
Cas9 and gRNA, which disrupted the function of DNMT3B in
transfected iPS cells (75). Using the same hPSC lines and delivery method, Ding et al. compared the efficiencies of CRISPR/
Cas9 and TALENs for genome editing of iPS cells (76). They
observed that CRISPR/Cas9 was more efficient than TALENs
(76). However, it is still a long road to clinically applying

CRISPR/Cas9 for gene therapy. We must ensure the high specificity of CRISPR/Cas9 for target sites and eliminate possible offtarget mutations with negative effects. Careful selection of target
sites, delicate gRNA design and genome-wide search of potential off-target sites are mostly required.

CHALLENGES
Despite the great potential of CRISPR/Cas9 in genome editing,
there are some important issues that need to be addressed, such as
off-target mutations, PAM dependence, gRNA production and
delivery methods of CRISPR/Cas9.
Off-target mutations
Off-target mutations are one major concern about CRISPR/
Cas9-mediated genome editing. Compared with ZFNs and
TALENs, CRISPR/Cas9 presents relative high risk of off-target
mutations in human cells (27). Large genomes often contain
multiple DNA sequences that are identical or highly homologous
to target DNA sequences. Besides target DNA sequences,
CRISPR/Cas9 also cleaves these identical or highly homologous
DNA sequences, which leads to mutations at undesired sites,
called off-target mutations. Off-target mutations can result in
cell death or transformation. To reduce the cellular toxicity of
CRISPR/Cas9, more and more efforts are paid to eliminate the
off-target mutations of CRISPR/Cas9 (26,27,29,77). To ensure
the specificity of CRISPR/Cas9, it is better to select the target
sites with the fewest off-target sites and mismatches between
gRNA and its complementary sequence. Xiao et al. recently
developed a flexible searching tool CasOT, which could identify
potential off-target sites across whole genomes (77). The dosage
of CRISPR/Cas9 is another factor affecting off-target mutations
and should be carefully controlled (29,78). Methylation of target
DNA sequences appeared not to affect the specificity of
CRISPR/Cas9 (29). Additionally, converting Cas9 into
nickase can help to reduce off-target mutations, while maintaining the efficiency of on-target cleavage implemented by
CRISPR/Cas9 (26).
PAM dependence
Theoretically, CRISPR/Cas9 can be applied to any DNA
sequence through engineered programmable gRNA. However,
the specificity of CRISPR/Cas9 requires a 2 5 nt PAM
sequence locating at immediately downstream of the target
sequence, besides gRNA/target sequence complementarity
(21). The identified PAM sequences vary among different
Cas9 orthologs, such as NGG PAM from Streptococcus pyogenes (21,79), NGGNG and NNAGAAW PAM from Streptococcus thermophiles (22,80,81) and NNNNGATT PAM from
Neisseria meningitidis (36,82). Recently, Hsu et al. reported a
NAG PAM, which had only 20% efficiency of NGG PAM
for guiding DNA cleavage (29). On the one hand, the PAMdependent manner of CRISPR/Cas9-mediated DNA cleavage
constrains the frequencies of targetable sites in genomes. For instance, it is possible to find a target site per 8 nt for NGG PAM
and NAG PAM, while per 32 and 256 nt for NGGNG PAM
and NNAGAAW PAM. On the other hand, PAM dependence

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

disrupting transcription-related functional sites, CRISPR/Cas9

can regulate the transcription of specific genes. However, this
process is irreversible due to permanent DNA modifications.
Recently, a modified CRISPR/Cas9 system named CRISPR
inference (CRISPRi) is develped for RNA-guided transcription
regulation (58 60). Qi et al. generated a catalytically defective
Cas9 (dCas9) mutant without nucleases activity. dCas9 was
co-expressed with gRNA to form a recognition complex,
which could interfere with transcriptional elongation, RNA
polymerase and transcription factor binding (60). With two
gRNA targeting, respectively, a red fluorescent protein (RFP)
gene and a green fluorescent protein (GFP) gene, Qi et al.
observed that CRISPRi could simultaneously repress the expression of RFP and GFP without crosstalk in Escherichia coli (60).
However, the degree of gene expression repression achieved by
CRISPRi was modest in mammalian cells (60). Gilbert et al.
fused repressive or activating effector domains to dCas9,
which together with gRNA could implement precise and stable
transcriptional control of target genes, including transcription
repression and activation (59). Chen et al. illustrated the performance of CRISPRi for individually or simultaneously regulating the transcription of multiple genes (58). CRISPRi
provides a novel highly specific tool for switching gene expression without genetically altering target DNA sequence.

R43

R44

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

also increases the specificity of CRISPR/Cas9. The off-target

mutations of CRISPR/Cas9 requiring long PAM should be less
than that of CRISPR/Cas9 requiring short PAM.

(2013KJXX-51) and the Fundamental Research Funds for the

Central Universities.

REFERENCES
gRNA production

Delivery methods
Questions also remain regarding the delivery methods of
CRISPR/Cas9 into organisms. DNA and RNA injection-based
techniques are used for CRISPR/Cas9 delivery, such as injection
of plasmids expressing Cas9 and gRNA (30) and injection of
CRISPR components as RNA (43,44). The efficiencies of delivery methods depend on the types of target cells and tissues. More
attentions should be paid to develop novel robust delivery
methods for CRISPR/Cas9.

CONCLUSION
Genome editing is initially applied to Drosophila melanogaster
(84,85), and rapidly extends to a broad range of organisms. An
ideal genome editing tool should have simple, efficient and
low-cost assembly of nucleases that can target any site without
off-target mutations in genomes. CRISPR/Cas9 has the potential
to become a reliable and facile genome editing tool, after addressing some issues. Benefiting from the simplicity and adaptability
of CRISPR/Cas9, it opens the door for revealing gene function in
biology and correcting gene defects in diseases. Further studies
are necessary to explore the characteristic and improve the performance of CRISPR/Cas9, especially the specificity, off-target
effects and delivery methods of CRISPR/Cas9. For instance,
recent genome-wide deeply sequencing results will be helpful
for selecting suitable target sites and designing highly specific
gRNA.
Conflict of Interest statement. None declared.

FUNDING
The study was supported by National Natural Scientific Fund of
China (81102086), Science and Technology Research and Development Program of in Shaanxi Province of China

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

gRNA production is another important issue for CRISPR/Cas9mediated genome editing. Due to extensive posttranscriptional
processing and modification of mRNA transcribed by RNA
polymerase II, it is currently difficult to apply RNA polymerase
II for gRNA production. RNA polymerase III, U3 and U6
snRNA promoters are currently used to produce gRNA in vivo.
However, U3 and U6 snRNA genes are ubiquitously expressed
housekeeping genes, which cannot be used to generate tissueand cell-specific gRNA (83). The lack of commercially available
RNA polymerase III also limits the application of U3- and
U6-based gRNA production. Gao et al. designed an artificial
gene RGR, the transcribed mRNA of which contained desired
gRNA and ribozyme sequences at both ends of gRNA (83).
After self-catalyzed cleavage, mature gRNA were produced
and successfully induced sequence-specific cleavage in vitro
and in yeast (83).

1. Banga, S.S. and Boyd, J.B. (1992) Oligonucleotide-directed site-specific

mutagenesis in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 89,
1735 1739.
2. Gloor, G.B., Nassif, N.A., Johnson-Schlitz, D.M., Preston, C.R. and Engels,
W.R. (1991) Targeted gene replacement in Drosophila via P
element-induced gap repair. Science, 253, 11101117.
3. Rong, Y.S. and Golic, K.G. (2000) Gene targeting by homologous
recombination in Drosophila. Science, 288, 20132018.
4. Gong, W.J. and Golic, K.G. (2003) Ends-out, or replacement, gene targeting
in Drosophila. Proc. Natl. Acad. Sci. USA, 100, 25562561.
5. Bhakta, M.S., Henry, I.M., Ousterout, D.G., Das, K.T., Lockwood, S.H.,
Meckler, J.F., Wallen, M.C., Zykovich, A., Yu, Y., Leo, H. et al. (2013)
Highly active zinc-finger nucleases by extended modular assembly. Genome
Res., 23, 530 538.
6. Kim, S., Lee, M.J., Kim, H., Kang, M. and Kim, J.S. (2011) Preassembled
zinc-finger arrays for rapid construction of ZFNs. Nat. Methods, 8, 7.
7. Gonzalez, B., Schwimmer, L.J., Fuller, R.P., Ye, Y., Asawapornmongkol, L.
and Barbas, C.F. 3rd (2010) Modular system for the construction of
zinc-finger libraries and proteins. Nat. Protoc., 5, 791810.
8. Kim, Y.G., Cha, J. and Chandrasegaran, S. (1996) Hybrid restriction
enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci.
USA, 93, 11561160.
9. Cathomen, T. and Joung, J.K. (2008) Zinc-finger nucleases: the next
generation emerges. Mol. Ther., 16, 12001207.
10. Townsend, J.A., Wright, D.A., Winfrey, R.J., Fu, F., Maeder, M.L., Joung,
J.K. and Voytas, D.F. (2009) High-frequency modification of plant genes
using engineered zinc-finger nucleases. Nature, 459, 442 445.
11. Wood, A.J., Lo, T.W., Zeitler, B., Pickle, C.S., Ralston, E.J., Lee, A.H.,
Amora, R., Miller, J.C., Leung, E., Meng, X. et al. (2011) Targeted genome
editing across species using ZFNs and TALENs. Science, 333, 307.
12. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye,
T., Nickstadt, A. and Bonas, U. (2009) Breaking the code of DNA binding
specificity of TAL-type III effectors. Science, 326, 1509 1512.
13. Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher governs DNA
recognition by TAL effectors. Science, 326, 1501.
14. Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu, J.K., Shi, Y. and
Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by
TAL effectors. Science, 335, 720 723.
15. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C.,
Baller, J.A., Somia, N.V., Bogdanove, A.J. and Voytas, D.F. (2011) Efficient
design and assembly of custom TALEN and other TAL effector-based
constructs for DNA targeting. Nucleic Acids Res., 39, e82.
16. Reyon, D., Tsai, S.Q., Khayter, C., Foden, J.A., Sander, J.D. and Joung, J.K.
(2012) FLASH assembly of TALENs for high-throughput genome editing.
Nat. Biotechnol., 30, 460465.
17. Briggs, A.W., Rios, X., Chari, R., Yang, L., Zhang, F., Mali, P. and Church,
G.M. (2012) Iterative capped assembly: rapid and scalable synthesis of
repeat-module DNA such as TAL effectors from individual monomers.
Nucleic Acids Res., 40, e117.
18. Schmid-Burgk, J.L., Schmidt, T., Kaiser, V., Honing, K. and Hornung, V.
(2013) A ligation-independent cloning technique for high-throughput
assembly of transcription activator-like effector genes. Nat. Biotechnol., 31,
76 81.
19. Kim, Y., Kweon, J., Kim, A., Chon, J.K., Yoo, J.Y., Kim, H.J., Kim, S., Lee,
C., Jeong, E., Chung, E. et al. (2013) A library of TAL effector nucleases
spanning the human genome. Nat. Biotechnol., 31, 251258.
20. Pennisi, E. (2013) The CRISPR craze. Science, 341, 833836.
21. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and
Charpentier, E. (2012) A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science, 337, 816821.
22. Gasiunas, G., Barrangou, R., Horvath, P. and Siksnys, V. (2012)
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage
for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA, 109,
E2579E2586.
23. Grissa, I., Vergnaud, G. and Pourcel, C. (2007) The CRISPRdb database and
tools to display CRISPRs and to generate dictionaries of spacers and repeats.
BMC Bioinform., 8, 172.

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

46. Wang, Y., Li, Z., Xu, J., Zeng, B., Ling, L., You, L., Chen, Y., Huang, Y. and
Tan, A. (2013) The CRISPR/Cas system mediates efficient genome
engineering in Bombyx mori. Cell Res., 23, 1414 1416.
47. Jiang, W., Bikard, D., Cox, D., Zhang, F. and Marraffini, L.A. (2013)
RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat.
Biotechnol., 31, 233239.
48. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J. and Church, G.M.
(2013) Genome engineering in Saccharomyces cerevisiae using
CRISPR-Cas systems. Nucleic Acids Res., 41, 4336 4343.
49. Xie, K. and Yang, Y. (2013) RNA-guided genome editing in plants using a
CRISPR-Cas system. Mol. Plant, 6, 1975 1983.
50. Upadhyay, S.K., Kumar, J., Alok, A. and Tuli, R. (2013) RNA-guided
genome editing for target gene mutations in wheat. G3 (Bethesda), 3,
22332238.
51. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J.D. and Kamoun, S. (2013)
Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9
RNA-guided endonuclease. Nat. Biotechnol., 31, 691693.
52. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J.,
Xi, J.J., Qiu, J.L. et al. (2013) Targeted genome modification of crop plants
using a CRISPR-Cas system. Nat. Biotechnol., 31, 686688.
53. Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J.,
Church, G.M. and Sheen, J. (2013) Multiplex and homologous
recombination-mediated genome editing in Arabidopsis and Nicotiana
benthamiana using guide RNA and Cas9. Nat. Biotechnol., 31, 688691.
54. Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D.L., Wei, P., Cao, F., Zhu, S.,
Zhang, F., Mao, Y. et al. (2013) Efficient genome editing in plants using a
CRISPR/Cas system. Cell Res., 23, 1229 1232.
55. Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F. and Zhu, J.K. (2013)
Application of the CRISPR-Cas system for efficient genome engineering in
plants. Mol. Plant, 6, 20082011.
56. Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H.
and Qu, L.J. (2013) Targeted mutagenesis in rice using CRISPR-Cas system.
Cell Res., 23, 12331236.
57. Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B. and Weeks, D.P. (2013)
Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene
modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res.,
41, e188.
58. Cheng, A.W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T.W.,
Rangarajan, S., Shivalila, C.S., Dadon, D.B. and Jaenisch, R. (2013)
Multiplexed activation of endogenous genes by CRISPR-on, an
RNA-guided transcriptional activator system. Cell Res., 23, 1163 1171.
59. Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E.,
Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A. et al.
(2013) CRISPR-mediated modular RNA-guided regulation of transcription
in eukaryotes. Cell, 154, 442 451.
60. Qi, L.S., Larson, M.H., Gilbert, L.A., Doudna, J.A., Weissman, J.S., Arkin,
A.P. and Lim, W.A. (2013) Repurposing CRISPR as an RNA-guided
platform for sequence-specific control of gene expression. Cell, 152,
11731183.
61. Urnov, F.D., Miller, J.C., Lee, Y.L., Beausejour, C.M., Rock, J.M.,
Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D. and Holmes,
M.C. (2005) Highly efficient endogenous human gene correction using
designed zinc-finger nucleases. Nature, 435, 646 651.
62. Li, H., Haurigot, V., Doyon, Y., Li, T., Wong, S.Y., Bhagwat, A.S., Malani,
N., Anguela, X.M., Sharma, R., Ivanciu, L. et al. (2011) In vivo genome
editing restores haemostasis in a mouse model of haemophilia. Nature, 475,
217 221.
63. Zou, J., Mali, P., Huang, X., Dowey, S.N. and Cheng, L. (2011) Site-specific
gene correction of a point mutation in human iPS cells derived from an adult
patient with sickle cell disease. Blood, 118, 45994608.
64. Sebastiano, V., Maeder, M.L., Angstman, J.F., Haddad, B., Khayter, C., Yeo,
D.T., Goodwin, M.J., Hawkins, J.S., Ramirez, C.L., Batista, L.F. et al. (2011)
In situ genetic correction of the sickle cell anemia mutation in human
induced pluripotent stem cells using engineered zinc finger nucleases. Stem
Cells, 29, 17171726.
65. Yusa, K., Rashid, S.T., Strick-Marchand, H., Varela, I., Liu, P.Q., Paschon,
D.E., Miranda, E., Ordonez, A., Hannan, N.R., Rouhani, F.J. et al. (2011)
Targeted gene correction of alpha1-antitrypsin deficiency in induced
pluripotent stem cells. Nature, 478, 391 394.
66. Soldner, F., Laganiere, J., Cheng, A.W., Hockemeyer, D., Gao, Q.,
Alagappan, R., Khurana, V., Golbe, L.I., Myers, R.H., Lindquist, S. et al.
(2011) Generation of isogenic pluripotent stem cells differing exclusively at
two early onset Parkinson point mutations. Cell, 146, 318 331.

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

24. Haft, D.H., Selengut, J., Mongodin, E.F. and Nelson, K.E. (2005) A guild of
45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas
subtypes exist in prokaryotic genomes. PLoS Comput. Biol., 1, e60.
25. Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J., Charpentier, E.,
Horvath, P., Moineau, S., Mojica, F.J., Wolf, Y.I., Yakunin, A.F. et al. (2011)
Evolution and classification of the CRISPR-Cas systems. Nat. Rev.
Microbiol., 9, 467477.
26. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu,
X., Jiang, W., Marraffini, L.A. et al. (2013) Multiplex genome engineering
using CRISPR/Cas systems. Science, 339, 819 823.
27. Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K. and
Sander, J.D. (2013) High-frequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells. Nat. Biotechnol., 31, 822 826.
28. Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S.,
Yang, L. and Church, G.M. (2013) CAS9 transcriptional activators for target
specificity screening and paired nickases for cooperative genome
engineering. Nat. Biotechnol., 31, 833838.
29. Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S.,
Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O. et al. (2013) DNA
targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol., 31,
827832.
30. Gratz, S.J., Cummings, A.M., Nguyen, J.N., Hamm, D.C., Donohue, L.K.,
Harrison, M.M., Wildonger, J. and OConnor-Giles, K.M. (2013) Genome
engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.
Genetics, 194, 1029 1035.
31. Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F.
and Jaenisch, R. (2013) One-step generation of mice carrying mutations in
multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153,
910918.
32. Yang, H., Wang, H., Shivalila, C.S., Cheng, A.W., Shi, L. and Jaenisch, R.
(2013) One-step generation of mice carrying reporter and conditional alleles
by CRISPR/Cas-mediated genome engineering. Cell, 154, 1370 1379.
33. Jinek, M., East, A., Cheng, A., Lin, S., Ma, E. and Doudna, J. (2013)
RNA-programmed genome editing in human cells. Elife, 2, e00471.
34. Cho, S.W., Kim, S., Kim, J.M. and Kim, J.S. (2013) Targeted genome
engineering in human cells with the Cas9 RNA-guided endonuclease. Nat.
Biotechnol., 31, 230 232.
35. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville,
J.E. and Church, G.M. (2013) RNA-guided human genome engineering via
Cas9. Science, 339, 823 826.
36. Hou, Z., Zhang, Y., Propson, N.E., Howden, S.E., Chu, L.F., Sontheimer,
E.J. and Thomson, J.A. (2013) Efficient genome engineering in human
pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl.
Acad. Sci. USA, 110, 1564415649.
37. Hruscha, A., Krawitz, P., Rechenberg, A., Heinrich, V., Hecht, J., Haass, C.
and Schmid, B. (2013) Efficient CRISPR/Cas9 genome editing with low
off-target effects in zebrafish. Development, 140, 49824987.
38. Auer, T.O., Duroure, K., De Cian, A., Concordet, J.P. and Del Bene, F.
(2014) Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by
homology-independent DNA repair. Genome Res., 24, 142153.
39. Jao, L.E., Wente, S.R. and Chen, W. (2013) Efficient multiplex biallelic
zebrafish genome editing using a CRISPR nuclease system. Proc. Natl.
Acad. Sci. USA, 110, 1390413909.
40. Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.W. and Xi, J.J.
(2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish
embryos. Cell Res., 23, 465 472.
41. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D.,
Peterson, R.T., Yeh, J.R. and Joung, J.K. (2013) Efficient genome editing in
zebrafish using a CRISPR-Cas system. Nat. Biotechnol., 31, 227229.
42. Shen, B., Zhang, J., Wu, H., Wang, J., Ma, K., Li, Z., Zhang, X., Zhang, P. and
Huang, X. (2013) Generation of gene-modified mice via Cas9/
RNA-mediated gene targeting. Cell Res., 23, 720 723.
43. Bassett, A.R., Tibbit, C., Ponting, C.P. and Liu, J.L. (2013) Highly efficient
targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell
Rep., 4, 220 228.
44. Yu, Z., Ren, M., Wang, Z., Zhang, B., Rong, Y.S., Jiao, R. and Gao, G. (2013)
Highly efficient genome modifications mediated by CRISPR/Cas9 in
Drosophila. Genetics, 195, 289 291.
45. Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaiacovo, M.P., Church, G.M.
and Calarco, J.A. (2013) Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nat. Methods, 10, 741743.

R45

R46

Human Molecular Genetics, 2014, Vol. 23, Review Issue 1

76. Ding, Q., Regan, S.N., Xia, Y., Oostrom, L.A., Cowan, C.A. and Musunuru,
K. (2013) Enhanced efficiency of human pluripotent stem cell genome
editing through replacing TALENs with CRISPRs. Cell Stem Cell, 12,
393394.
77. Xiao, A., Cheng, Z., Kong, L., Zhu, Z., Lin, S., Gao, G. and Zhang, B. (2014)
CasOT: a genome-wide Cas9/gRNA off-target searching tool.
Bioinformatics. [Epub ahead of print].
78. Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A. and Liu, D.R.
(2013) High-throughput profiling of off-target DNA cleavage reveals
RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol., 31,
839843.
79. Deltcheva, E., Chylinski, K., Sharma, C.M., Gonzales, K., Chao, Y., Pirzada,
Z.A., Eckert, M.R., Vogel, J. and Charpentier, E. (2011) CRISPR RNA
maturation by trans-encoded small RNA and host factor RNase III. Nature,
471, 602 607.
80. Garneau, J.E., Dupuis, M.E., Villion, M., Romero, D.A., Barrangou, R.,
Boyaval, P., Fremaux, C., Horvath, P., Magadan, A.H. and Moineau, S.
(2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage
and plasmid DNA. Nature, 468, 67 71.
81. Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P. and
Siksnys, V. (2013) crRNA and tracrRNA guide Cas9-mediated DNA
interference in Streptococcus thermophilus. RNA Biol., 10, 841851.
82. Zhang, Y., Heidrich, N., Ampattu, B.J., Gunderson, C.W., Seifert, H.S.,
Schoen, C., Vogel, J. and Sontheimer, E.J. (2013) Processing-independent
CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol.
Cell, 50, 488 503.
83. Gao, Y. and Zhao, Y. (2013) Self-processing of ribozyme-flanked RNAs into
guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J.
Integr. Plant Biol. [Epub ahead of print].
84. Bibikova, M., Golic, M., Golic, K.G. and Carroll, D. (2002) Targeted
chromosomal cleavage and mutagenesis in Drosophila using zinc-finger
nucleases. Genetics, 161, 1169 1175.
85. Bibikova, M., Beumer, K., Trautman, J.K. and Carroll, D. (2003) Enhancing
gene targeting with designed zinc finger nucleases. Science, 300, 764.

Downloaded from http://hmg.oxfordjournals.org/ by guest on March 8, 2016

67. Perez, E.E., Wang, J., Miller, J.C., Jouvenot, Y., Kim, K.A., Liu, O., Wang,
N., Lee, G., Bartsevich, V.V., Lee, Y.L. et al. (2008) Establishment of HIV-1
resistance in CD4+ T cells by genome editing using zinc-finger nucleases.
Nat. Biotechnol., 26, 808816.
68. Holt, N., Wang, J., Kim, K., Friedman, G., Wang, X., Taupin, V., Crooks,
G.M., Kohn, D.B., Gregory, P.D., Holmes, M.C. et al. (2010) Human
hematopoietic stem/progenitor cells modified by zinc-finger nucleases
targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol., 28, 839 847.
69. Voit, R.A., McMahon, M.A., Sawyer, S.L. and Porteus, M.H. (2013)
Generation of an HIV resistant T-cell line by targeted stacking of
restriction factors. Mol. Ther., 21, 786795.
70. Torikai, H., Reik, A., Liu, P.Q., Zhou, Y., Zhang, L., Maiti, S., Huls, H.,
Miller, J.C., Kebriaei, P., Rabinovitch, B. et al. (2012) A foundation for
universal T-cell based immunotherapy: T cells engineered to express a
CD19-specific chimeric-antigen-receptor and eliminate expression of
endogenous TCR. Blood, 119, 5697 5705.
71. Provasi, E., Genovese, P., Lombardo, A., Magnani, Z., Liu, P.Q., Reik, A.,
Chu, V., Paschon, D.E., Zhang, L., Kuball, J. et al. (2012) Editing T cell
specificity towards leukemia by zinc finger nucleases and lentiviral gene
transfer. Nat. Med., 18, 807 815.
72. Ebina, H., Misawa, N., Kanemura, Y. and Koyanagi, Y. (2013) Harnessing
the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep., 3, 2510.
73. Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., DeKelver,
R.C., Katibah, G.E., Amora, R., Boydston, E.A., Zeitler, B. et al. (2009)
Efficient targeting of expressed and silent genes in human ESCs and iPSCs
using zinc-finger nucleases. Nat. Biotechnol., 27, 851857.
74. Ding, Q., Lee, Y.K., Schaefer, E.A., Peters, D.T., Veres, A., Kim, K.,
Kuperwasser, N., Motola, D.L., Meissner, T.B., Hendriks, W.T. et al. (2013)
A TALEN genome-editing system for generating human stem cell-based
disease models. Cell Stem Cell, 12, 238 251.
75. Horii, T., Tamura, D., Morita, S., Kimura, M. and Hatada, I. (2013)
Generation of an ICF syndrome model by efficient genome editing of human
induced pluripotent stem cells using the CRISPR system. Int. J. Mol. Sci., 14,
1977419781.