- Email: [email protected]
Plant genome engineering in full bloom
Techniques & Applications
Special Issue: Systems Biology
Plant genome engineering in full bloom Jorge Lozano-Juste1,2,3 and Sean R. Cutler1,2,3 1
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA 3 Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA 2
The recent development of tools for precise editing of user-specified sequences is rapidly changing the landscape for plant genetics and biotechnology. It is now possible to target mutations and regulatory proteins to specific sites in a genome using zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), or the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we provide an update of recent developments in CRISPR/Cas9 technology and highlight online resources that will help biologists adopt new genome-editing tools.
Genome engineering Genome editing is broadly defined as the ability to make tailored changes to a genome’s sequence. Biologists working with a select group of model organisms have been able to make targeted genetic changes by homologous recombination for many years, but reliable systems for higher plants have eluded the efforts of many laboratories. This is now changing, owing to technologies that trace their routes to work in the late 1990s when researchers developed new methods for designing synthetic zinc-finger DNA-binding proteins from modular building blocks. These synthetic proteins were shown to regulate the transcription of specific sequences in vivo when fused to transcriptional activation or repression domains [1]. Parallel work designing chimeric zinc fingers fused to the FokI nuclease established the feasibility of creating doublestrand breaks (DSBs) at tailored sites [2]. The development of synthetic zinc-finger nucleases (ZFNs) launched genome editing in its current incarnation and is changing the way geneticists study gene function. The power of this approach emerges from the ability of DSBs to recruit DNA repair factors acting in either the nonhomologous end joining (NHEJ) or homologous recombination (HR) pathways. NHEJ repairs the breaks, but is imprecise and can create mutations at and around the DSB. When the HR machinery repairs the DSB, sequences with homology flanking the DSB (including exogenously supplied Corresponding author: Cutler, S.R. ([email protected]). Keywords: genome engineering; ZFNs; TALENs; CRISPR; Cas9; plant. 1360-1385/$ – see front matter. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tplants.2014.02.014
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sequences) can be incorporated into the genome. DSBs can therefore be leveraged to make changes at defined sites; however, intrinsic differences between the relative roles of HR and NHEJ can affect the rates of HR observed. ZFNs are costly to make and substantial efforts are often invested to create the high-affinity DNA-binding domains that are necessary for genome engineering; Transcription activator-like effectors (TALEs) address this problem because they require less improvement after construction. TALEs are DNA-binding proteins produced by plant pathogens that are secreted into plant cells where they directly recognize DNA and alter transcription of endogenous loci. TALEs comprise multiple copies of a 33–35-amino acid repeat. DNA recognition by TALEs is determined by two variable amino acids within the repeat. This recognition code has been used to create artificial TALENs [3,4], which have a higher likelihood of functioning in vivo after construction than ZFNs [5]. However, like ZFNs, TALENs require the time-consuming assembly of small building blocks to create synthetic DNA-binding proteins. This time-consuming step is not required with the recently developed CRISPR/Cas9 system, which provides a technically simpler system for genome editing. Here we review the CRISPR/Cas9 system and examine recent innovations that address its off-target effects. We additionally highlight online tools that improve and simplify construct design (Box 1). The CRISPR/Cas9 blast A major advance in genome engineering occurred in 2012, with the discovery of the CRISPR/Cas9 system [6]. In this bacterial antiviral and transcriptional regulatory system, a complex of two small RNAs – the CRISPR-RNA (crRNA) and the trans-activating crRNA (tracrRNA) – directs the nuclease (Cas9) to a specific DNA sequence complementary to the crRNA [6], similar to the way that small interfering RNAs (siRNAs) guide argonaute proteins to specific RNA sequences during gene silencing. Binding of these RNAs to Cas9 requires specific sequences and secondary structures in the RNA. The two RNA components have been simplified into a single element, the guide-RNA (gRNA), which is transcribed from a cassette containing a target sequence defined by the user [6] (Figure 1). Since this seminal work, the system has been successfully used for genome engineering in humans, zebrafish, Drosophila, mice, nematodes, bacteria, yeast, and plants [7]. The gRNA comprises two components: a 20-nucleotide (nt) sequence complementary to the target DNA that is
Techniques & Applications
Trends in Plant Science May 2014, Vol. 19, No. 5
Box 1. Genome editing on the net
Excitingly, HR-dependent gene replacement was reported in two of the above studies [11,17]. These recent successes demonstrate the generality of CRISPR/Cas9 mutagenesis across kingdoms.
The ready availability of materials for genome engineering has helped to spawn the rapid growth of these technologies. The nonprofit plasmid repository Addgene (http://www.addgene.org) contains dozens of plasmids for creating ZFNs, TALENs, or CRISPR/ Cas9 for many organisms, including CRISPR/Cas9 plasmids deposited by the Kamoun laboratory [19]. The Sheen and Gao laboratories constructed a codon-optimized Cas9 for Arabidopsis thaliana and rice (Oryza sativa), respectively, along with gRNA cassettes, which are available on request [11,17]. In addition to obtaining the basic building blocks, adopters of these technologies will benefit from online design tools. ZiFit (http://zifit.partners.org/ZiFiT/) helps to construct gRNAs, TALENs, and ZFNs targeting the sequence of interest [8]. The CRISPR Design Tool from Feng Zhang’s laboratory (http://crispr.mit.edu/) helps design gRNA sequences that are predicted to minimize off-target mutations. This tool includes the prediction of paired gRNAs for double-nickase experiments and helps with the discovery of off-targets genome wide. The current version includes Arabidopsis but will soon be updated with other plant genomes. Another tool including Arabidopsis, Brachypodium, and rice genomes is E-CRISP (http://e-crisp-test.dkfz.de/E-CRISP/ index.html), which also permits the finding of paired gRNAs and offtargets. Additionally, the CRISPR-PLANT Database (http://www.genome.arizona.edu/crispr/index.html) is an online tool that includes more plant genomes. With the prediction of gRNA libraries able to target more than 85% of the transcriptional units of seven different plant species, this is currently by far the most extensive gRNA database dedicated to plants [30]. Online discussion groups have also arisen that will undoubtedly assist new users of these technologies (https://groups.google.com/forum/#!forum/talengineering; https://groups.google.com/forum/#!forum/crispr).
embedded in a larger, approximately 100-nt RNA sequence that forms the necessary secondary structure required for Cas9 binding (Figure 1) [6,8]. The target sequence must end in 50 -NGG-30 [the protospacer adjacent motif (PAM)] for recognition by the Cas9–gRNA complex. After target recognition, Cas9 cleaves DNA 3 nt upstream of the PAM on the complementary strand and 3–8 nt upstream on the noncomplementary strand (Figure 1). The PAM places a modest constraint on target site sequences; however it appears that 50 -NAG-30 may also work [9]. These minimal sequence requirements ensure that the vast majority of genes will be suitable for CRISPR/Cas9 targeting. The CRISPR/Cas9 system has useful features. It allows one to program target recognition using synthetic gRNAs instead of synthetic DNA-binding domains, which makes it easier to implement than ZFNs and TALENs. Another benefit is that higher-order mutants can be constructed in a single step [10–12]. This is possible because Cas9 binds whatever cellular gRNAs are present; coexpression of different gRNAs creates complexes that target multiple sites. The Cas9/gRNA system can also be used to target chimeric Cas9 proteins to defined sites for the purpose of regulating gene expression. This is achieved by fusing regulatory domains to a mutant Cas9 nuclease domain (dCas9) that cannot introduce DSBs at target sites [13] (Figure 1). Most studies to date have been conducted in animal systems, but CRISPR/Cas9-mediated mutagenesis was recently demonstrated in Arabidopsis, tobacco, sorghum, rice, and wheat, proving that this technique is applicable to both dicot and monocot plants [11,14–19]. The mutation rates obtained with the CRISPR/Cas9 system are comparable with those observed with ZFNs and TALENs, being particularly high in stably transformed plants [14].
CRISPR/Cas9 limitations All technologies have limitations and an obvious problem with genome engineering relates to off-target effects. Notably, the Cas9–gRNA complex can tolerate multiple mismatches between the target and the gRNA [9,20,21], with mismatches in the 50 region of the gRNA being better tolerated than those at other locations (Figure 1). However, alterations at the PAM motif or of gRNA length dramatically affect the ability of the CRISPR/Cas9 system to generate DSBs [9]. These observations suggest that judicious design may help reduce off-target effects. Off-target activity is affected by the ratio of gRNA to Cas9 protein produced by target cells. Unsurprisingly, increasing gRNA levels decreases off-target effects [20–22]. For this reason the use of a strong constitutive promoter (i.e., 35S) to drive Cas9 expression may not be ideal in plants, although this remains to be investigated systematically. Additionally, off-target effects can be reduced using a double-nicking strategy in which paired gRNAs are used in combination with the nickase Cas9D10A (Cas9n), which is mutated in one of the nuclease motifs (Figure 1). Off-target effects have been reduced by 50–15 000-fold using this approach [22]. The use of truncated-gRNAs (tru-gRNAs) can also reduce off-target effects by approximately 5000-fold [23]. With only 17–18-nt homology to the target sequence, trugRNAs tolerate fewer mismatches but retain the mutation efficiency that 20-nt gRNAs possess. Additionally, unlike paired nickases, tru-gRNAs can be used for the control of gene expression. Off-target activity varies by orders of magnitude between different target sequences and organisms, so this issue needs to be explicitly addressed in plant systems. Analyses of multiple alleles and tests for cosegregation of phenotypes and target mutations as well as complementation experiments can be used to demonstrate causal relationships between a mutation and phenotype. Additionally, off-target mutations can be identified and eliminated from analyses by re-sequencing, which will become increasing feasible for large numbers of mutant strains as genome re-sequencing costs continue to drop. Concluding remarks: what’s next? Genome editing is creating new opportunities for crop design; in particular, it may allow the generation of nontransgenic genetically altered crops, although it remains unresolved whether plants modified by genome engineering will be classified as genetically modified organisms (GMOs). To date, Agrobacterium-mediated transformation has been the system of choice for delivering DNA into plant cells, although new viral vectors may facilitate donor DNA delivery for gene editing [24]. Inducible gene expression in combination with the strategies discussed here can yield a powerful new control system [25], but remain untested in plants. Improving Cas9 and engineering better gRNA scaffolds may reduce off-target effects. Cas9 proteins from different 285
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Trends in Plant Science May 2014, Vol. 19, No. 5
(A) Cas9
Guide RNA
+
Nuclease domains
Target complementary sequence
+
+
RuvC Target DNA sequence
–
HNH
Mismatch tolerance
(B)
CRISPR/Cas9 Genome eding
G G N NAA G G
(C)
CRISPR/Cas9n Double nicking
Cas9n (D10A) (Nickase)
Cas9n (D10A) (Nickase)
(D)
CRISPR/dCas9 Transcriponal regulaon
dCas9
Repressor Acvator
(Inacve)
Promoter region
Target gene
TRENDS in Plant Science
Figure 1. The CRISPR/Cas9 system. (A) The guide-RNA (gRNA) includes the 20-nucleotide (nt) target complementary sequence that will guide the Cas9 nuclease toward its target. The gradient of mismatch tolerance between the gRNA and the target DNA is highlighted from red to white indicating high (red) to low specificity (white). The two different nuclease domains that enable double-strand breaks (DSBs) (RuvC and HNH) are depicted as white arrowheads. (B) CRISPR/Cas9-mediated genome editing. The gRNA interacts with Cas9 and directs it to the complementary target DNA sequence and trinucleotide NA/GG protospacer adjacent motif (PAM) (in orange). The nuclease domains of the Cas9 produce DSBs 3 nt upstream of the PAM sequence on the complementary strand and 3–8 nt upstream of the PAM on the noncomplementary strand. After DSB production, endogenous DNA repair machinery can be exploited to modify the target sequence in multiple ways. (C) Double nicking to reduce off-target effects. A pair of gRNAs recruits a ‘nickase’ mutant Cas9 (Cas9n) to the target site. The targeted Cas9n proteins each produce a nick at their target sites, which generates a DSB and recruits the non-homologous end joining (NHEJ) and homologous recombination (HR) machinery. Using this approach, similar on-target mutations rates are observed but off-target rates are reduced. (D) Controlling transcription using the CRISPR/Cas9 system. A catalytically inactivated version of Cas9 (dCAS9) is fused to either a transcriptional activator or repressor domain. This enables targeted modulation of transcript levels by localizing the gRNA–dCas9–effector domain complex to regulatory regions. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeat; Cas9, CRISPR-associated protein 9.
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Techniques & Applications organisms have been characterized [26] and, with differing PAM requirements, these new types of Cas9 enlarge the toolbox for genome editing, allowing the user to choose the most appropriate Cas9–gRNA combination. Additionally, orthogonal gene targeting in which separate gRNAs are targeted to distinct Cas9 proteins provides the opportunity to regulate gene expression and gene editing in a single plant [27]. Furthermore, whole-genome gRNA libraries will allow the generation of mutant lines not yet available in public seed banks, along with genome-wide CRISPR/Cas9 knockout (GeCKO) libraries for forward genetics [28,29]. Genome engineering tools will help to advance faster our understanding of not only model plants but also important crops with unanticipated relevance in biotechnology. Acknowledgments This work was supported by the National Science Foundation (Integrative Organismal Systems: 0820508) to S.R.C. The authors thank Laetitia Poidevin and Inge Verstraeten for comments on the manuscript. They also thank Laurent Poidevin for graphic design. They apologize to colleagues whose work could not be cited owing to space limitations.
References 1 Beerli, R.R. et al. (1998) Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. U.S.A. 95, 14628–14633 2 Kim, Y-G. et al. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 1156–1160 3 Boch, J. et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 4 Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 5 Beumer, K.J. et al. (2013) Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila. G3 (Bethesda) 3, 1717–1725 6 Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 7 Puchta, H. and Fauser, F. (2013) Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J. http:// dx.doi.org/10.1111/tpj.12338 8 Mali, P. et al. (2013) RNA-guided human genome engineering via Cas9. Science 339, 823–826 9 Mali, P. et al. (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 10 Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823
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11 Li, J-F. et al. (2013) Multiplex and homologous recombinationmediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 12 Wang, H. et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 13 Gilbert, L.A. et al. (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 14 Feng, Z. et al. (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229–1232 15 Jiang, W. et al. (2013) Demonstration of CRISPR/Cas9/sgRNAmediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. http://dx.doi.org/10.1093/nar/ gkt780 16 Miao, J. et al. (2013) Targeted mutagenesis in rice using CRISPR–Cas system. Cell Res. 23, 1233–1236 17 Shan, Q. et al. (2013) Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 18 Xie, K. and Yang, Y. (2013) RNA-guided genome editing in plants using a CRISPR–Cas system. Mol. Plant 6, 1975–1983 19 Nekrasov, V. et al. (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 691–693 20 Fu, Y. et al. (2013) High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 21 Pattanayak, V. et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 22 Ran, F.A. et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 23 Fu, Y. et al. (2014) Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. http://dx.doi.org/10.1038/ nbt.2808 24 Baltes, N.J. et al. (2014) DNA replicons for plant genome engineering. Plant Cell http://dx.doi.org/10.1105/tpc.113.119792 25 Konermann, S. et al. (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 26 Fonfara, I. et al. (2013) Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. http://dx.doi.org/10.1093/ nar/gkt1074 27 Esvelt, K.M. et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods http://dx.doi.org/10.1038/ nmeth.2681 28 Shalem, O. et al. (2014) Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 29 Wang, T. et al. (2014) Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 30 Xie, K. et al. (2014) Genome-wide prediction of highly specific guide RNA spacers for the CRISPR–Cas9 mediated genome editing in model plants and major crops. Mol. Plant http://dx.doi.org/10.1093/mp/ ssu009
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Special Issue: Systems Biology
Plant genome engineering in full bloom Jorge Lozano-Juste1,2,3 and Sean R. Cutler1,2,3 1
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA 3 Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA 2
The recent development of tools for precise editing of user-specified sequences is rapidly changing the landscape for plant genetics and biotechnology. It is now possible to target mutations and regulatory proteins to specific sites in a genome using zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), or the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we provide an update of recent developments in CRISPR/Cas9 technology and highlight online resources that will help biologists adopt new genome-editing tools.
Genome engineering Genome editing is broadly defined as the ability to make tailored changes to a genome’s sequence. Biologists working with a select group of model organisms have been able to make targeted genetic changes by homologous recombination for many years, but reliable systems for higher plants have eluded the efforts of many laboratories. This is now changing, owing to technologies that trace their routes to work in the late 1990s when researchers developed new methods for designing synthetic zinc-finger DNA-binding proteins from modular building blocks. These synthetic proteins were shown to regulate the transcription of specific sequences in vivo when fused to transcriptional activation or repression domains [1]. Parallel work designing chimeric zinc fingers fused to the FokI nuclease established the feasibility of creating doublestrand breaks (DSBs) at tailored sites [2]. The development of synthetic zinc-finger nucleases (ZFNs) launched genome editing in its current incarnation and is changing the way geneticists study gene function. The power of this approach emerges from the ability of DSBs to recruit DNA repair factors acting in either the nonhomologous end joining (NHEJ) or homologous recombination (HR) pathways. NHEJ repairs the breaks, but is imprecise and can create mutations at and around the DSB. When the HR machinery repairs the DSB, sequences with homology flanking the DSB (including exogenously supplied Corresponding author: Cutler, S.R. ([email protected]). Keywords: genome engineering; ZFNs; TALENs; CRISPR; Cas9; plant. 1360-1385/$ – see front matter. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tplants.2014.02.014
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sequences) can be incorporated into the genome. DSBs can therefore be leveraged to make changes at defined sites; however, intrinsic differences between the relative roles of HR and NHEJ can affect the rates of HR observed. ZFNs are costly to make and substantial efforts are often invested to create the high-affinity DNA-binding domains that are necessary for genome engineering; Transcription activator-like effectors (TALEs) address this problem because they require less improvement after construction. TALEs are DNA-binding proteins produced by plant pathogens that are secreted into plant cells where they directly recognize DNA and alter transcription of endogenous loci. TALEs comprise multiple copies of a 33–35-amino acid repeat. DNA recognition by TALEs is determined by two variable amino acids within the repeat. This recognition code has been used to create artificial TALENs [3,4], which have a higher likelihood of functioning in vivo after construction than ZFNs [5]. However, like ZFNs, TALENs require the time-consuming assembly of small building blocks to create synthetic DNA-binding proteins. This time-consuming step is not required with the recently developed CRISPR/Cas9 system, which provides a technically simpler system for genome editing. Here we review the CRISPR/Cas9 system and examine recent innovations that address its off-target effects. We additionally highlight online tools that improve and simplify construct design (Box 1). The CRISPR/Cas9 blast A major advance in genome engineering occurred in 2012, with the discovery of the CRISPR/Cas9 system [6]. In this bacterial antiviral and transcriptional regulatory system, a complex of two small RNAs – the CRISPR-RNA (crRNA) and the trans-activating crRNA (tracrRNA) – directs the nuclease (Cas9) to a specific DNA sequence complementary to the crRNA [6], similar to the way that small interfering RNAs (siRNAs) guide argonaute proteins to specific RNA sequences during gene silencing. Binding of these RNAs to Cas9 requires specific sequences and secondary structures in the RNA. The two RNA components have been simplified into a single element, the guide-RNA (gRNA), which is transcribed from a cassette containing a target sequence defined by the user [6] (Figure 1). Since this seminal work, the system has been successfully used for genome engineering in humans, zebrafish, Drosophila, mice, nematodes, bacteria, yeast, and plants [7]. The gRNA comprises two components: a 20-nucleotide (nt) sequence complementary to the target DNA that is
Techniques & Applications
Trends in Plant Science May 2014, Vol. 19, No. 5
Box 1. Genome editing on the net
Excitingly, HR-dependent gene replacement was reported in two of the above studies [11,17]. These recent successes demonstrate the generality of CRISPR/Cas9 mutagenesis across kingdoms.
The ready availability of materials for genome engineering has helped to spawn the rapid growth of these technologies. The nonprofit plasmid repository Addgene (http://www.addgene.org) contains dozens of plasmids for creating ZFNs, TALENs, or CRISPR/ Cas9 for many organisms, including CRISPR/Cas9 plasmids deposited by the Kamoun laboratory [19]. The Sheen and Gao laboratories constructed a codon-optimized Cas9 for Arabidopsis thaliana and rice (Oryza sativa), respectively, along with gRNA cassettes, which are available on request [11,17]. In addition to obtaining the basic building blocks, adopters of these technologies will benefit from online design tools. ZiFit (http://zifit.partners.org/ZiFiT/) helps to construct gRNAs, TALENs, and ZFNs targeting the sequence of interest [8]. The CRISPR Design Tool from Feng Zhang’s laboratory (http://crispr.mit.edu/) helps design gRNA sequences that are predicted to minimize off-target mutations. This tool includes the prediction of paired gRNAs for double-nickase experiments and helps with the discovery of off-targets genome wide. The current version includes Arabidopsis but will soon be updated with other plant genomes. Another tool including Arabidopsis, Brachypodium, and rice genomes is E-CRISP (http://e-crisp-test.dkfz.de/E-CRISP/ index.html), which also permits the finding of paired gRNAs and offtargets. Additionally, the CRISPR-PLANT Database (http://www.genome.arizona.edu/crispr/index.html) is an online tool that includes more plant genomes. With the prediction of gRNA libraries able to target more than 85% of the transcriptional units of seven different plant species, this is currently by far the most extensive gRNA database dedicated to plants [30]. Online discussion groups have also arisen that will undoubtedly assist new users of these technologies (https://groups.google.com/forum/#!forum/talengineering; https://groups.google.com/forum/#!forum/crispr).
embedded in a larger, approximately 100-nt RNA sequence that forms the necessary secondary structure required for Cas9 binding (Figure 1) [6,8]. The target sequence must end in 50 -NGG-30 [the protospacer adjacent motif (PAM)] for recognition by the Cas9–gRNA complex. After target recognition, Cas9 cleaves DNA 3 nt upstream of the PAM on the complementary strand and 3–8 nt upstream on the noncomplementary strand (Figure 1). The PAM places a modest constraint on target site sequences; however it appears that 50 -NAG-30 may also work [9]. These minimal sequence requirements ensure that the vast majority of genes will be suitable for CRISPR/Cas9 targeting. The CRISPR/Cas9 system has useful features. It allows one to program target recognition using synthetic gRNAs instead of synthetic DNA-binding domains, which makes it easier to implement than ZFNs and TALENs. Another benefit is that higher-order mutants can be constructed in a single step [10–12]. This is possible because Cas9 binds whatever cellular gRNAs are present; coexpression of different gRNAs creates complexes that target multiple sites. The Cas9/gRNA system can also be used to target chimeric Cas9 proteins to defined sites for the purpose of regulating gene expression. This is achieved by fusing regulatory domains to a mutant Cas9 nuclease domain (dCas9) that cannot introduce DSBs at target sites [13] (Figure 1). Most studies to date have been conducted in animal systems, but CRISPR/Cas9-mediated mutagenesis was recently demonstrated in Arabidopsis, tobacco, sorghum, rice, and wheat, proving that this technique is applicable to both dicot and monocot plants [11,14–19]. The mutation rates obtained with the CRISPR/Cas9 system are comparable with those observed with ZFNs and TALENs, being particularly high in stably transformed plants [14].
CRISPR/Cas9 limitations All technologies have limitations and an obvious problem with genome engineering relates to off-target effects. Notably, the Cas9–gRNA complex can tolerate multiple mismatches between the target and the gRNA [9,20,21], with mismatches in the 50 region of the gRNA being better tolerated than those at other locations (Figure 1). However, alterations at the PAM motif or of gRNA length dramatically affect the ability of the CRISPR/Cas9 system to generate DSBs [9]. These observations suggest that judicious design may help reduce off-target effects. Off-target activity is affected by the ratio of gRNA to Cas9 protein produced by target cells. Unsurprisingly, increasing gRNA levels decreases off-target effects [20–22]. For this reason the use of a strong constitutive promoter (i.e., 35S) to drive Cas9 expression may not be ideal in plants, although this remains to be investigated systematically. Additionally, off-target effects can be reduced using a double-nicking strategy in which paired gRNAs are used in combination with the nickase Cas9D10A (Cas9n), which is mutated in one of the nuclease motifs (Figure 1). Off-target effects have been reduced by 50–15 000-fold using this approach [22]. The use of truncated-gRNAs (tru-gRNAs) can also reduce off-target effects by approximately 5000-fold [23]. With only 17–18-nt homology to the target sequence, trugRNAs tolerate fewer mismatches but retain the mutation efficiency that 20-nt gRNAs possess. Additionally, unlike paired nickases, tru-gRNAs can be used for the control of gene expression. Off-target activity varies by orders of magnitude between different target sequences and organisms, so this issue needs to be explicitly addressed in plant systems. Analyses of multiple alleles and tests for cosegregation of phenotypes and target mutations as well as complementation experiments can be used to demonstrate causal relationships between a mutation and phenotype. Additionally, off-target mutations can be identified and eliminated from analyses by re-sequencing, which will become increasing feasible for large numbers of mutant strains as genome re-sequencing costs continue to drop. Concluding remarks: what’s next? Genome editing is creating new opportunities for crop design; in particular, it may allow the generation of nontransgenic genetically altered crops, although it remains unresolved whether plants modified by genome engineering will be classified as genetically modified organisms (GMOs). To date, Agrobacterium-mediated transformation has been the system of choice for delivering DNA into plant cells, although new viral vectors may facilitate donor DNA delivery for gene editing [24]. Inducible gene expression in combination with the strategies discussed here can yield a powerful new control system [25], but remain untested in plants. Improving Cas9 and engineering better gRNA scaffolds may reduce off-target effects. Cas9 proteins from different 285
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Trends in Plant Science May 2014, Vol. 19, No. 5
(A) Cas9
Guide RNA
+
Nuclease domains
Target complementary sequence
+
+
RuvC Target DNA sequence
–
HNH
Mismatch tolerance
(B)
CRISPR/Cas9 Genome eding
G G N NAA G G
(C)
CRISPR/Cas9n Double nicking
Cas9n (D10A) (Nickase)
Cas9n (D10A) (Nickase)
(D)
CRISPR/dCas9 Transcriponal regulaon
dCas9
Repressor Acvator
(Inacve)
Promoter region
Target gene
TRENDS in Plant Science
Figure 1. The CRISPR/Cas9 system. (A) The guide-RNA (gRNA) includes the 20-nucleotide (nt) target complementary sequence that will guide the Cas9 nuclease toward its target. The gradient of mismatch tolerance between the gRNA and the target DNA is highlighted from red to white indicating high (red) to low specificity (white). The two different nuclease domains that enable double-strand breaks (DSBs) (RuvC and HNH) are depicted as white arrowheads. (B) CRISPR/Cas9-mediated genome editing. The gRNA interacts with Cas9 and directs it to the complementary target DNA sequence and trinucleotide NA/GG protospacer adjacent motif (PAM) (in orange). The nuclease domains of the Cas9 produce DSBs 3 nt upstream of the PAM sequence on the complementary strand and 3–8 nt upstream of the PAM on the noncomplementary strand. After DSB production, endogenous DNA repair machinery can be exploited to modify the target sequence in multiple ways. (C) Double nicking to reduce off-target effects. A pair of gRNAs recruits a ‘nickase’ mutant Cas9 (Cas9n) to the target site. The targeted Cas9n proteins each produce a nick at their target sites, which generates a DSB and recruits the non-homologous end joining (NHEJ) and homologous recombination (HR) machinery. Using this approach, similar on-target mutations rates are observed but off-target rates are reduced. (D) Controlling transcription using the CRISPR/Cas9 system. A catalytically inactivated version of Cas9 (dCAS9) is fused to either a transcriptional activator or repressor domain. This enables targeted modulation of transcript levels by localizing the gRNA–dCas9–effector domain complex to regulatory regions. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeat; Cas9, CRISPR-associated protein 9.
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Techniques & Applications organisms have been characterized [26] and, with differing PAM requirements, these new types of Cas9 enlarge the toolbox for genome editing, allowing the user to choose the most appropriate Cas9–gRNA combination. Additionally, orthogonal gene targeting in which separate gRNAs are targeted to distinct Cas9 proteins provides the opportunity to regulate gene expression and gene editing in a single plant [27]. Furthermore, whole-genome gRNA libraries will allow the generation of mutant lines not yet available in public seed banks, along with genome-wide CRISPR/Cas9 knockout (GeCKO) libraries for forward genetics [28,29]. Genome engineering tools will help to advance faster our understanding of not only model plants but also important crops with unanticipated relevance in biotechnology. Acknowledgments This work was supported by the National Science Foundation (Integrative Organismal Systems: 0820508) to S.R.C. The authors thank Laetitia Poidevin and Inge Verstraeten for comments on the manuscript. They also thank Laurent Poidevin for graphic design. They apologize to colleagues whose work could not be cited owing to space limitations.
References 1 Beerli, R.R. et al. (1998) Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. U.S.A. 95, 14628–14633 2 Kim, Y-G. et al. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 1156–1160 3 Boch, J. et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 4 Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 5 Beumer, K.J. et al. (2013) Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila. G3 (Bethesda) 3, 1717–1725 6 Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 7 Puchta, H. and Fauser, F. (2013) Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J. http:// dx.doi.org/10.1111/tpj.12338 8 Mali, P. et al. (2013) RNA-guided human genome engineering via Cas9. Science 339, 823–826 9 Mali, P. et al. (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 10 Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823
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11 Li, J-F. et al. (2013) Multiplex and homologous recombinationmediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 12 Wang, H. et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 13 Gilbert, L.A. et al. (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 14 Feng, Z. et al. (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229–1232 15 Jiang, W. et al. (2013) Demonstration of CRISPR/Cas9/sgRNAmediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. http://dx.doi.org/10.1093/nar/ gkt780 16 Miao, J. et al. (2013) Targeted mutagenesis in rice using CRISPR–Cas system. Cell Res. 23, 1233–1236 17 Shan, Q. et al. (2013) Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 18 Xie, K. and Yang, Y. (2013) RNA-guided genome editing in plants using a CRISPR–Cas system. Mol. Plant 6, 1975–1983 19 Nekrasov, V. et al. (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 691–693 20 Fu, Y. et al. (2013) High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 21 Pattanayak, V. et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 22 Ran, F.A. et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 23 Fu, Y. et al. (2014) Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. http://dx.doi.org/10.1038/ nbt.2808 24 Baltes, N.J. et al. (2014) DNA replicons for plant genome engineering. Plant Cell http://dx.doi.org/10.1105/tpc.113.119792 25 Konermann, S. et al. (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 26 Fonfara, I. et al. (2013) Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. http://dx.doi.org/10.1093/ nar/gkt1074 27 Esvelt, K.M. et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods http://dx.doi.org/10.1038/ nmeth.2681 28 Shalem, O. et al. (2014) Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 29 Wang, T. et al. (2014) Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 30 Xie, K. et al. (2014) Genome-wide prediction of highly specific guide RNA spacers for the CRISPR–Cas9 mediated genome editing in model plants and major crops. Mol. Plant http://dx.doi.org/10.1093/mp/ ssu009
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