Cas9 for plant genome editing

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promoter sequence of a gene are coupled with this dCas9- activator/repressor ... Transcriptional activation is also possible with dCas9 ..... Fusing dCas9 to p300,.
Plant Cell Rep DOI 10.1007/s00299-016-1985-z

REVIEW

CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects Joseph W. Paul III1 • Yiping Qi1

Received: 30 January 2016 / Accepted: 12 April 2016 Ó Springer-Verlag Berlin Heidelberg 2016

Abstract The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development. Keywords Genome editing  Plant biotechnology  Transcriptional regulation  Gene targeting  Synthetic biology  CRISPR  Cas9

Introduction Methods for rapidly and efficiently editing plant genomes are powerful tools for basic research and crop improvement. Tools such as zinc finger nucleases (ZFNs) (Carroll 2011; Urnov et al. 2010) and TAL effector nucleases (TALENs) (Christian et al. 2010; Li et al. 2011; Miller et al. 2010) can be custom engineered to create precise Communicated by T. Cardi. & Yiping Qi [email protected] 1

Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA

double strand breaks (DSBs) at targeted DNA sites, creating mutations or allowing for insertion of DNA sequences. More recently, a new technology (Jinek et al. 2012), which relies on an RNA-guided nuclease, was re-engineered to efficiently edit genomes across a variety of eukaryotes and has become an important tool for genome editing in plants. This system is derived from clustered regularly interspaced short palindromic repeats (CRISPR) and the RNA-guided double-stranded DNA binding protein Cas9 that comprise type II effector systems. In nature, it functions as an adaptive immune apparatus in prokaryotes composed of two RNA molecules and a single protein nuclease (Wiedenheft et al. 2012). Genome editing in eukaryotes can be accomplished by ectopically introducing two CRISPR/Cas9 components into the same cell: a guide RNA (gRNA) and the Cas9 nuclease. The gRNA is a chimera of the naturally occurring CRISPR RNA (crRNA), which is complementary to the target DNA sequence, and the transactivating CRISPR RNA (tracrRNA), which forms a structural bridge between the crRNA and Cas9. Cas9 is an RNA-guided DNA nuclease containing two twinned, catalytically active nuclease domains (NHN and RuvC). Cas9 is ferried to a DNA sequence by complexing with a scaffold structure in the gRNA (Fig. 1a). The gRNA spacer region can then anneal to a complementary DNA sequence with Cas9, and create a DSB, so long as the target sequence neighbors a protospacer adjacent motif (PAM). Cas9 orthologs may differ in their requirements for gRNA scaffold structures and PAM sequences in order to efficiently create a DSB at the target site. The most commonly used Cas9 is derived from Streptococous pyogenes and requires an NGG (N, any nucleotide; G, guanine) PAM sequence for DNA targeting. These DSBs are repaired by evolutionarily conserved DNA repair pathways. The predominant pathway to repair DSBs

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Plant Cell Rep Fig. 1 a CRISPR/Cas9 complexes target specific genomic sequences to create double strand breaks adjacent to the PAM site. b The error-prone non-homologous end-joining (NHEJ) DSB repair pathway can be harnessed by CRISPR/ Cas9 to introduce pre-mature stop codons in the coding sequence of a gene. c Homologous recombination (HR) is a less efficient, but useful pathway for repairing DSBs in the presence of a DNA template so that precise genome editing can be achieved

is non-homologous end-joining (NHEJ), which can be error-prone resulting in the introduction of insertion or deletion (indel) at the target site (Fig. 1b). These mutations can disrupt genes by creating a premature stop codon in the open reading frame, leading to nonsense-mediated decay of the transcript. Alternatively, homology-directed repair (HDR) can result in the introduction of new sequences when a DNA template with homology to the target sequence is provided (Fig. 1c).

A highly flexible genome editing tool for plants Reprogramming of CRISPR/Cas9 to cleave a designated DNA sequence was first accomplished in vitro in 2012 (Jinek et al. 2012) followed by successful multiplex genome editing in mammalian cells (Cong et al. 2013; Mali et al. 2013a) in early 2013. Several months later, the first application of CRISPR/Cas9 was reported in plants (Li et al. 2013a; Nekrasov et al. 2013; Shan et al. 2013), which spurred wide and rapid application to many other plant species (Fig. 2). Consistent with other genome editing tools, NHEJ is most easily harnessed to create mutant alleles. The efficiency of Cas9 editing varies depending on plant species, genomic loci targeted, expression levels of gRNA and Cas9, among other factors (Bortesi and Fischer 2014).

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Fig. 2 Number of genes reported edited by CRISPR/Cas9 to date, by plant species. Publications reporting these were gathered using PubMed and searching the terms ‘‘crispr’’ and ‘‘plant’’. ‘‘Tobacco’’ includes Nicotiana tabacum and Nicotiana benthamiana

CRISPR/Cas9 can also be used to edit genes with HDR, albeit with far less efficiency than NHEJ. However, harnessing the power of HDR can be used to introduce new DNA sequences into targeted genomic loci with precision. The first demonstration of HDR-edited plants with CRISPR/Cas9 was the introduction of a kanamycin resistance cassette into the ADH1 gene locus in Arabidopsis thaliana (Schiml et al. 2014). Agrobacterium was used to transform plants with a single transfer DNA (T-DNA)

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construct containing Cas9 and a gRNA adjacent to the resistance cassette which was flanked by 674 base pairs of homology to the ADH1 locus. Using a combination of PCR-based and Southern blot analysis, the authors confirmed the HDR events in T1 lines and further demonstrated successful germinal transmission in some lines. In a recent paper, HDR in plants is reported using a similar method to introduce a kanamycin resistance cassette upstream of a 35S promoter in between the ANT1 promoter and open reading frame (ORF) in tomatoes (Cermak et al. 2015). Overexpression of ANT1 results in high accumulation of anthocyanin and produces a purple plant phenotype providing a convenient screening method to identify successfully targeted plants. CRISPR/Cas9 performed comparably to TALENs to mediate gene targeting in this assay. Two recent reports described CRISPR/Cas9-based gene targeting in maize (Svitashev et al. 2015) and soybean (Li et al. 2015). In the former, a slightly different approach involved introducing Cas9, gRNA, and a repair template using Agrobacterium transformation and particle bombardment. Particle bombardment successfully delivered the CRISPR/Cas9 reagents for gene targeting, albeit at low frequency (*4 %). Though, particle bombardment can lead to undesired DNA rearrangements and tandem or truncated insertions (Pawlowski and Somers 1998). Soybean plants were also subjected to particle bombardment to introduce Cas9, gRNA, and a repair template. Similarly, the authors found low efficiency of gene targeting that was heritable in subsequent generations. More recently, HDR was reported in rice, where herbicide resistance trait was engineered by specific modification of the endogenous ALS gene (Sun et al. 2016). Importantly, the authors found that providing HDR donors in the form of both the plasmid and free double-stranded DNA drastically improved HDR efficiency. By contrast, single-stranded oligonucleotides cannot effectively function as HDR donor templates in their system. However, in Arabidopsis and flax (Linum usitatissimum) protoplasts, it has recently been shown that CRISPR/Cas9 with single-stranded oligonucleotide templates can precisely edit the genome at a higher efficiency than single-stranded oligonucleotide alone (Sauer et al. 2016).

Expression and delivery Artificial expression of CRISPR/Cas9 systems in plants takes advantage of existing plant biotechnologies. Most systems express Cas9 and gRNA(s) separately: promoters such as the cauliflower mosaic virus derived 35S promoter drives strong expression of Cas9 by RNA polymerase II while small nuclear RNA promoters, such as the U6 or U3

promoter, facilitates RNA polymerase III transcription of the gRNA with defined initiation and termination sites. One caveat of this system is the need for separate elements (promoter and terminator) to express multiple gRNAs. In this regard, Agrobacterium/T-DNA and viral systems inherently limit the number of gRNAs that can be expressed due to maximum length of the DNA component to be assembled or packaged. One solution harnesses ribozyme sequences adjacent to gRNA sequences in the transcribed RNA to precisely cleave out a functional gRNA (Gao and Zhao 2014; Nissim et al. 2014). Two ribozymes, hammerhead (HH) type and one from the hepatitis delta virus (HDV), are cloned downstream and upstream of the gRNA, respectively, to accomplish this. Moreover, a theoretical construct could express both Cas9 and gRNAs flanked by ribozyme sequences on the same vector in a compact transcript that would be easier to deliver and allow for better spatiotemporal control of both components of the CRISPR/Cas9 system. In principle, polycistronic transcripts can give rise to multiple gRNAs to render multiplex capabilities of Cas9. Because this action occurs independent of the promoter, either constitutive or inducible polymerase II promoters can be used for the expression of gRNAs in such a system. Another system expresses gRNAs in the context of tRNA ‘‘leader’’ and ‘‘trailer’’ sequences (which are specifically cleaved by RNases P and Z, respectively) in a polycistronic RNA from an RNA polymerase III promoter to achieve a similar effect in plants (Xie et al. 2015). The most common delivery method for CRISPR/Cas9 in plants is Agrobacterium-based transformation (reviewed in (Gelvin 2003) which introduces T-DNA directly into the plant genome by a type IV secretion mechanism. Cas9 and gRNA expression cassettes can be easily cloned into a specialized ‘‘tumor inducing’’ or Ti plasmid, transformed into Agrobacterium, and applied to plants. This method was the first employed for CRISPR/Cas9 genome editing in plants; first during 2013 (Li et al. 2013a; Nekrasov et al. 2013; Shan et al. 2013) and in many instances after. However, other methods have recently been shown to provide enhanced control over CRISPR/Cas9 delivery, particularly in live plants where HDR-based gene targeting can be difficult. Several viral approaches have been recently deployed into plants to combats some of these challenges of transgene delivery (Ali et al. 2015; Baltes et al. 2014, 2015; Yin et al. 2015). However, these viral systems are chiefly limited by the size of their nucleic acid cargo—most geminivirus genomes are *3 kb while the Cas9 coding sequence (from S. pyogenes) alone is *4.2 kb. Thus, viruses are more practical for delivering gRNA(s) to plants stably expressing Cas9 but could still be a valuable tool for rapid reverse genetic screening in plants.

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Additionally, a DNA-free method of genome editing with CRISPR/Cas9 was recently reported that directly introduces preassembled gRNA:Cas9 complexes into plant protoplasts of Arabidopsis, tobacco, and rice (Woo et al. 2015). This method was also extended into lettuce (Lactuca sativa) to mutate the BIN2 gene in protoplasts which can be regenerated into calli, and later, fully grown plants, with a reported mutation efficiency of 46 %. These mutations occurred on both alleles of the target gene that were transmitted to the next generation. This system is particularly advantageous because it may not fall under the regulatory scrutiny faced by transgenic crops, as they are devoid of transgenes encoding CRISPR/Cas9 machinery. With the goal of avoiding regulation, however, the system is only suitable to the creation of indel mutations and not for gene targeting which would nonetheless require an exogenous DNA template.

Multiplex genome engineering Methods for rapid assembly of plasmids encoding the components of genome editing systems have been quickly adapted for CRISPR/Cas9 in plants. In late 2014, the first toolkit to streamline the construction of CRISPR/Cas9 plasmids for Agrobacterium-based expression in plants emerged (Xing et al. 2014). GoldenGate cloning (Engler et al. 2009) is used to combine PCR amplified gRNA targeting sequences into a single vector. The final vector contains a maize-codon optimized Cas9 sequence behind an ubiquitin promoter and two gRNA behind U6 promoters. The authors demonstrate that this tool can efficiently edit DNA in maize and Arabidopsis plants. In the case of Arabidopsis, mutations were heritable to the T2 generation. A second toolkit was published early in 2015 (Ma et al. 2015b) that also features a PCR-based protocol with complete, one-step construction of all components by GoldenGate cloning or Gibson Assembly (Gibson et al. 2009). Notably, the kit can be used for high-efficiency mutagenesis in rice and heritable mutations in Arabidopsis. A CRISPR/Cas9 toolbox that does not require PCR for construction and can also be used for transcriptional regulation with dCas9 fusion was also developed and demonstrated in plants (Lowder et al. 2015). Two recent publications demonstrated the assembly of multiplex CRISPR/Cas9 systems with conventional cloning approaches. In one case, six gRNA-expression cassettes were constructed into a single vector with Cas9 in three steps (Zhang et al. 2015b). Although less efficient, this approach allowed for the simultaneous editing of six target sites. In another study, an assembly system based on an isocaudamer technique with compatible restriction enzymes was also developed (Wang et al. 2015a). Three

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genes were simultaneously targeted in rice with the system. Although laborious and time-consuming, this system, in theory, allows for unlimited stacking of gRNA expression cassettes.

Transcriptional regulation Targeted regulation of transcription in plants with artificial transcriptional factors is very useful in basic and translational science (Petolino and Davies 2013). The CRISPR/ Cas9 predecessors ZFNs and TALENs are derived from protein domains whose endogenous role in nature is to affect gene expression by binding to specific genomic loci. Cas9’s DNA binding function can be exploited to influence gene expression by deactivating its nuclease function (by point mutations in its two catalytic domains, HNH and RuvC) and fusing general transcriptional activators or repressors to its C-terminus. When gRNAs targeting the promoter sequence of a gene are coupled with this dCas9activator/repressor, the accessibility of the gene to RNA polymerase is altered. The RNA-dependent programmability of Cas9 makes it an attractive choice for this purpose over zinc finger- or TALE-based transcription factors that require separate assembly of a new protein for each target site. In each case, however, programmable transcriptional activators overcome many issues associated with ectopic overexpression or RNAi knockdown by allowing for the study of gene gain or loss-of-function in its genomic context. Moreover, CRISPR/Cas9 endows the ability to multiplex gene activation or repression. As a repressor of transcription, programming dCas9 to a site of transcription is sufficient to block RNA polymerase processivity (termed CRISPR interference or CRISPRi) (Qi et al. 2013a). In mammalian cells, CRISPRi can reduce gene expression levels, and is comparable to RNAi (i.e. siRNA, shRNA), but also has several key advantages, such as low cytotoxicity, high variability of function between organisms and experimental conditions due to reliance on cytoplasmic machinery for silencing, and less off-target effects. Off-target silencing effects are particularly important for genome-scale screens and were shown to be minimal with the use of CRISPRi (Gilbert et al. 2014). In plants, robust silencing has been best shown with dCas9 fused to the SRDX repressor motif, a highly generalizable transcriptional repressor (Hiratsu et al. 2003), to achieve nearly 80 % silencing (Piatek et al. 2015) and up to 65 % silencing in our toolkit (Lowder et al. 2015). A critical question of repression is the impact of gRNA location on silencing efficiency. Based on a screen of known genes that suppress ricin toxicity in mammalian cells, a region of -50 to ?300 base pairs (bp) relative to the genes’ transcription start site (TSS) were delineated for the highest silencing

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efficiency by CRISPRi (Gilbert et al. 2014). Thorough investigation of this feature has not been described in plants for dCas9-SRDX, but could be identified in the future by high-throughput analysis in, for example, protoplasts. Transcriptional activation is also possible with dCas9 fusion proteins fused to a tetramer of Herpes Viral Protein 16 (termed VP64). When recruited to the promoter sequence of a target gene by a gRNA, dCas9-VP64 recruits general transcription factors to facilitate transcription in mammalian cells (Gilbert et al. 2014; Nissim et al. 2014). dCas9-VP64 (Lowder et al. 2015) and dCas9 fused to an EDLL domain (a plant-specific transcriptional activator Tiwari et al. 2012) have both been shown to induce robust activation of synthetic reporters and endogenous genomic loci. dCas9-VP64 could also overcome CpG methylation to activate the imprinted FIS2 gene in Arabidopsis (Lowder et al. 2015). In both reports of dCas9-mediated activation in plants, with the exception of the imprinted FIS2 gene, expression levels of endogenous genes were increased by up to ten-fold. Although higher levels of expression may be desirable to observe more pronounced phenotypes associated with the function of certain genes, these results clearly show that Cas9-based transcription factors can activate endogenous gene expression. An alternative route for activation involves coupling dCas9-VP64 to a modified gRNA containing MS2 stem loops (gRNA 2.0) to recruit MS2 coat protein fused to additional activators (Konermann et al. 2015; Zalatan et al. 2015). This method was shown to yield high activation (50–300 fold increase in expression) of target genes in cultured mammalian cells when dCas9-VP64 is paired with gRNA 2.0 and MS2VP64. An additional prospect for transcriptional regulation is the fusion of other activation domains derived from plant proteins (Li et al. 2013b). It could be advantageous to test these domains together to potentially achieve the synergistic activation that has been reported in mammalian systems (Konermann et al. 2015).

Defending against plant pathogens Geminiviruses are single-stranded circular DNA viruses that replicate within the plant cell as double-stranded DNA (Mansoor et al. 2006). They are major plant pathogens that destroy their host plants, leading to reduced crop yields worldwide (Moffat 1999). While other molecular approaches for generating geminivirus-resistant plants have been reported with limited success (Araga˜o and Faria 2009; Yang et al. 2014), this presents a highly translatable problem at the level of DNA that could also be confronted by CRISPR/Cas9. Given that geminiviruses use rolling-circle replication to copy DNA (Gutierrez 1999), DSBs generated

within the viral DNA sequence could fundamentally compromise this process. This technique has been recently used to create tobacco (Nicotiana benthamiana) with enhanced resistance (Ali et al. 2015; Baltes et al. 2015; Ji et al. 2015) to three different geminiviruses, beet severe curly top virus, bean yellow dwarf virus, and tomato yellow leaf curl virus. A key advantage of CRISPR/Cas9 for this purpose lies in multiplexed interference with the viral DNA which improves the rate and efficiency of mutagenesis and subsequently leads to the reduction of the geminivirus-mediated phenotypes. These findings were extended to Arabidopsis, where a comparable reduction in plant sensitivity to the virus was reported (Ji et al. 2015). These proof-of-principle studies provide a new avenue for applied research towards reprogramming CRISPR/Cas9 for plant defense against a variety of viruses that rely on DNA integrity at some point of their replication cycle. It will be necessary to analyze how effectively CRISPR/Cas9 could work to disable virus in relevant crop species that are normally susceptible to geminiviruses, such as tomato and cotton. With unique attention to the ssDNA nature of geminiviruses, the recently reported DNase H activity of Cas9 derived from Neisseria meningitidis (Zhang et al. 2015a) could be harnessed for even more efficient targeting of the viral genome.

Challenges in germline genome editing A low frequency of germline genome editing by CRISPR/ Cas9 is observed in certain plants, particularly the model organism Arabidopsis. When Arabidopsis plants are subjected to Agrobacterium transformation of CRISPR/Cas9 by floral dip, mutation efficiency of target sites can be high in somatic cells, by virtue of expression behind a ubiquitous promoter like 35S, but can be low in reproductive cells, limiting the likelihood that mutations will be inherited in the next generation. To solve this, promoters to express Cas9 in germline-specific cells were used to generate higher frequency of mutations in subsequent generations in Arabidopsis. Egg cell-specific promoters were used for expression of Cas9 to generate non-mosaic T1 mutants (Wang et al. 2015b). It was further demonstrated that by simply stacking two such promoters together, a much higher frequency of mutagenesis can be obtained. This can also be accomplished by expressing Cas9 driven by the SPOROCYTELESS promoter, which encodes a transcription factor present at high levels in sporpogenesis (Mao et al. 2015). Although mutations in the T1 generation are rare using this new expression cassette, T2 mutants are more abundant than from plants expressing Cas9 behind a ubiquitous promoter. Similarly, Cas9 expressed behind an alternate promoter, from the YAO gene (which is

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preferentially expressed in mitotic cells) can also stimulate germline genome editing at greater efficiencies. This generated an extremely high frequency of edited T1 plants versus a ubiquitous promoter (90 vs. 4.3 %) when targeting a genomic sequence. These results suggest that the change of promoter to drive Cas9 expression is a fruitful area to explore for generating more efficient germline mutations. Unlike Arabidopsis, genome-edited rice plants are typically obtained through tissue culture, where the totipotency of somatic cells is utilized. This results in high-frequency germline modification. Partially due to this reason, more genes have been targeted by CRISPR/Cas9 in rice than in any other plant species (Fig. 2).

Tipping the scale of DNA repair As mentioned, DNA repair of DSBs induced by CRISPR/ Cas9 proceeds through two pathways: NHEJ or HDR (Fig. 1). NHEJ is most useful for creating indels in exons, often resulting in premature stop codons. The size of indels varies but the blunt cutting activity of Cas9 typically generates 1 bp indels, which are more difficult to detect by methods other than DNA sequencing. For example, the predominant 1 bp indels generated by CRISPR/Cas9 were reported in rice (Ma et al. 2015b; Zhang et al. 2014) and Arabidopsis (Feng et al. 2014). The most immediate solution to this is to program gRNAs to target two adjacent sites to create larger deletions. Alternatively, it is possible to block or inhibit (by RNAi or orthogonal CRISPRi/ dCas9-based repression) components of the classical NHEJ pathway (e.g. Ku70, Ku80, Lig4) to promote microhomology-based repair which is an alternative NHEJ pathway. Promoting larger deletions with this strategy has been demonstrated in plants with ZFN and TALEN. For example, we previously showed that repair of ZFN-induced DNA DSBs in ku70 or lig4 mutant in Arabidopsis resulted in larger deletions (Qi et al. 2013b). More recently, a higher frequency of larger deletions ([10 bp) was generated by TALEN in rice when Lig4 is knocked out (Nishizawa-Yokoi et al. 2015). Similarly, DNA ligase may also be inhibited, by the small molecule Scr7 (Maruyama et al. 2015), to promote microhomology-based alternative NHEJ as demonstrated in mammalian cells. A major challenge in all eukaryotes, however, is harnessing HDR to introduce new DNA in genomes with both precision and accuracy. This is particularly important for creating improved crops. To overcome this problem, knocking out or transiently silencing critical components of the NHEJ pathway could enhance the efficiency of HDR (Qi et al. 2013b). Also, overexpression of yeast Rad54, an ATPase that modifies the topology of DNA to facilitate HDR, in Arabidopsis increases HDR efficiency to

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incorporate a new DNA template by one to two orders of magnitude (Shaked et al. 2005). A chemical screen in mammalian cells recently characterized compounds that could enhance CRISPR/Cas9 editing by HDR threefold for larger insertions (Yu et al. 2015). Another chemical compound, RS-1, was previously found to stimulate HDR by promoting active presynaptic filaments formed by RAD51 and single-stranded DNA in vitro and in human cells (Jayathilaka et al. 2008). More recently, RS-1 was shown to enhance HDR-mediated by CRISPR/Cas9 and TALEN in rabbits (Song et al. 2016). These small molecules, as well as Scr7 (Maruyama et al. 2015), might be applied to plant protoplasts or cell cultures to enhance HDR in the same way. Besides manipulation of DNA repair pathways, enhancing HDR donor accessibility is another strategy for promoting HDR. For example, donor delivery in the form of incoming transfer DNA (T-DNA) was found to be used more effectively for HDR than a donor that is chromosome-integrated (Puchta 1999; Puchta et al. 1996). By liberating a chromosome-integrated donor with nucleasebased excision, HDR frequency may reach 1 %. This method is called in planta gene targeting (Fauser et al. 2012). Further, HDR frequency seems to be positively correlated with the copy number of freely available donor molecules. It has been shown that when a donor DNA fragment was delivered by geminivirus-based replicons (which can replicate up to hundreds of copies in a cell), HDR was greatly enhanced in tobacco somatic cells (Baltes et al. 2014).

Cas9 off-target binding and cleavage As with all genome editing tools, specificity is an important consideration because off-target cleavage of DNA can result in unwanted mutations and chromosomal abnormalities. Cas9:gRNA complexes are able to cleave target DNA sequences even with several mismatches in the guide sequence, implying that these complexes are capable of cutting at other genomic sites (Fu et al. 2013; Hsu et al. 2013). Mismatches of guide sequences are better tolerated at the 50 end, distal to the PAM sequence. Initially, highthroughput profiling of Cas9 specificity in mammalian cells demonstrated that the full 20 bp gRNA sequences endow a high degree of specificity (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013), contradicting previous reports that only 7–12 bp adjacent to the PAM sequence are important for Cas9 specificity. It has been reported that truncated guide RNAs (less than 20 bp) confer improved specificity for genome editing by Cas9 (Fu et al. 2014), implying that a shorter sequence than initially described may serve as the actual ‘‘seed’’ for Cas9 targeting to DNA. A promising

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result in rabbits was recently reported, in which CRISPR/ Cas9 genome editing resulted in no detectable off-target effects (Honda et al. 2015). Inactivation of either of the two conserved nuclease domains of Cas9 generates a nickase, which is a nuclease that only cleaves one strand of the DNA double helix. These nicks alone are not usually prone to causing mutation; they are repaired by the base excision repair pathway. When paired together by two distinct gRNAs targeting adjacent genomic sequences, two nickases can generate a complete DSB with sticky ends (Mali et al. 2013b; Ran et al. 2013) (Fig. 3a). This system drastically increases the specificity of genome editing (by 50? fold over conventional Cas9) because it requires the recognition by two gRNAs, expanding the target sequence from 20 to 40 bp. Indeed, Cas9 nickases also provide increased protection from off-target cleavage (Cho et al. 2014). In Arabidopsis and rice, it was demonstrated that paired Cas9 nickases do not outperform the nuclease in promoting either NHEJ or HDR repair, but may reduce off-target effects (Fauser et al. 2014; Mikami et al. 2016). In an alternative system to improve specificity, dCas9 is fused to the nonspecific nuclease domain of FokI (Fig. 3b), as with ZFNs and TALENs, which must dimerize to become catalytically active (Guilinger et al. 2014; Tsai et al. 2014). This system also requires two Cas9:gRNA complexes to be recruited to adjacent genomic targets. In Arabidopsis, this system performs as expected in reducing off-target cleavage when

compared to Cas9, however with a large trade-off in efficiency (Aouida et al. 2015). In situations where a high frequency of mutations is not desired over precise mutagenesis or gene targeting, it may be desirable to pursue the Cas9 nickase or Cas9-FokI systems to take advantage of increased specificity. Further addressing the off-targeting issue, two recent reports described improved Cas9 nucleases with vastly improved specificity. In one case, a structure-guided approach to identify a positively charged groove in Cas9 that likely stabilizes the non-target DNA strand was employed (Slaymaker et al. 2016). The authors reasoned that neutralization of the groove (via alanine substitution of positively charged amino acids) could diminish this interaction leading to increased Cas9 reliance on the gRNA base pairing to the target strand for stability. In mammalian cell culture, two of these mutant Cas9s (eSpCas9) demonstrated comparable, albeit more variable, editing efficiency across multiple genomic targets and increased sensitivity to base mismatches between the gRNA and target DNA when compared to the wild-type (WT) Cas9. Using BLESS, a method to map DSBs genome-wide using next generation sequencing (Crosetto et al. 2013), eSpCas9 also greatly reduced off-target cutting across the genome. In a complementary approach, the Cas9 structure was also probed to find amino acid residues that stabilize the target DNA strand and the 50 end of the gRNA (distal to the PAM) via hydrogen bonding (Kleinstiver et al. 2016). By mutating four of these sites (which do not overlap with those altered by Slaymaker et al.), they created a Cas9 (SpCas9-HF1) that also retains the ability to efficiently edit target genomic loci and with higher sensitivity to mismatches between the gRNA and target DNA sequences. Similarly, using another high-throughput, genome-wide analysis of DSBs (Tsai et al. 2015), it was shown that the specificity of SpCas9-HF1 is drastically increased for its target site. While only analyzed in mammalian cells thus far, these improved Cas9 nucleases have the potential to function with equally improved specificity in plants. With respect to crop development, this could be a valuable tool for ensuring that CRISPR/Cas9-edited organisms are largely free of off-target mutations.

Expanding CRISPR/Cas9 function and versatility

Fig. 3 Paired Cas9 systems for improving targeting specificity. a The paired nickase system that relies on two Cas9s with a deactivating mutation in one of the two nuclease domains (D10A in the RuvC domain or H840A in the HNH domain) guided to adjacent DNA sites. b The paired dCas9-FokI system also requires two complexes to be guided to adjacent DNA sites

The versatility of CRISPR/Cas9 to modify the genome at many levels of its composition and structure led to the genome editing revolution currently underway. With regard to basic research in plant biology, the expansion of this tool will continue alongside its use as a standard tool for reverse genetics studies (Fig. 1). Gene targeting by HDR is a key aspect of CRISPR/Cas9 technology that must

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be improved to solidify its usefulness over other genome editing tools, like TALENs or ZFNs. In live plants, this could prove to be much more challenging than in protoplasts or cell culture, due to the feasibility of applying small molecules to promote HDR in cultured cells. Additionally, delivery of CRISPR/Cas9 transgenes also presents a greater challenge in many plants. Thus, methods for rapidly culturing plant protoplast, expression of CRISPR/ Cas9 and manipulation of specific DNA repair pathways, and regeneration of whole plants will be of great interest to the plant research community. Another largely unrealized CRISPR/Cas9 opportunity is the application of orthogonal Cas9 proteins from other organisms beyond Streptococcus pyogenes for genome engineering. Cas9 proteins from two microorganisms, Streptococcus thermophiles (St) and Neisseria meningitidis (Nm), function effectively to edit and activate specific genomic sequences in eukaryotic cells (Esvelt et al. 2013) (Fig. 4). Strikingly, the gRNA scaffolds and PAM requirements are distinct enough among the aforementioned Cas9s to not cross-react with non-specific gRNA (e.g. SpCas9 cannot cut using a gRNA with the Nm-specific scaffold and PAM). Hence, orthogonal Cas9-based synthetic transcriptional activator and repressor could be co-expressed in the

same plant cells for orthogonal transcriptional regulation of different genes (Fig. 4), enabling sophisticated transcriptome reprogramming. In plants, efficient genome editing with Cas9 derived from Staphylococcus aureus (Sa) and StCas9 has been reported (Steinert et al. 2015). In addition to orthogonal Cas9 proteins, repurposing of gRNA scaffolds to include additional elements, such as synthetic RNA binding modules to bind a variety of proteins, can be paired with dCas9 to accomplish simultaneous functions similar to those proposed for orthogonal Cas9s. This method was demonstrated using gRNAs with additional elements that could bind artificial transcriptional activators or repressors, thereby allowing simultaneous control of activation or repression of specific genomic loci (Zalatan et al. 2015). Together, these advancements represent a large advancement towards synthetic control over transcriptional programs by using CRISPR/Cas9. In plants, the realization of these tools for precise gene expression could allow for control of gene expression regulatory networks (Petolino and Davies 2013; Puchta 2016), and thus complex traits, that have only recently become possible to dissect with computational methods (Krouk et al. 2013). CRISPR/Cas9 can also be used to image specific genomic loci in situ using fluorescently tagged dCas9

Fig. 4 Additional applications of CRISPR/Cas9 (Clockwise). Cas9 derived from different organisms for orthogonal genome editing (Nm Neisseria meningitidis, Sa Staphylococcus aureus, Sp Streptococous pyogenes, St Streptococcus thermophiles). Chromatin remodeling by fusing a histone modifying enzyme (e.g. histone deacetylase) to

dCas9 and targeting to specific genomic loci with a gRNA. Visualization of genomic loci in live cells with dCas9 fused to GFP. Simultaneous and orthogonal transcriptional regulation by dCas9 derived from different organisms

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(Fig. 4). In mammalian cells, three orthogonal dCas9s fused to different fluorescent proteins could illuminate genomic loci at three distinct regions (Ma et al. 2015a). The fluorescent signals could also be used to calculate the difference between these genomic loci. This application could allow for visual observation of interacting DNA sequences, perhaps revealing enhancer regions in DNA that interact with active transcription initiation sites. Additionally, directing dCas9 to genomic loci with affinity tags could allow for co-immunoprecipitation (Co-IP) of chromatin and allow for close examination of chromatin modifications or chromatin-associated proteins near specific genomic loci (Fujita and Fujii 2013). Epigenome engineering is also now possible with CRISPR/Cas9 technology (Fig. 4). Fusing dCas9 to p300, a mammalian acetyltransferase, can catalyze the acetylation of histone H3 at lysine 27 at its target sites (Hilton et al. 2015). This mark decondenses associated DNA leading to increased gene activation that was reported at levels exceeding activation by dCas9-VP64 fusion proteins. However, activation is only the surface of this technology which could include dCas9 fused to a variety of histone post-translational effectors. It is conceivable that dCas9 fused to domains that can control deacetylation, methylation, or phosphorylation of histones could also be of use to influence gene expression in eukaryotic cells. In the future, it will be of interest to apply these tools and others to achieve greater control of plant epigenomes. Also on the horizon, the new type II CRISPR nuclease Cpf1 offers distinct advantages over Cas9 that could even further propel genome engineering. Cpf1 is slightly smaller than Cas9 and two of its organismal derivations (from Acidominococcus and Lachnospiraceae) were shown to be effective in mammalian cells (Zetsche et al. 2015) and may also be applicable to plants. Contrary to Cas9, Cpf1 contains three RuvC domains without an HNH domain. Cpf1 is guided by single gRNA (*42 bp as opposed to the *100 bp gRNAs for Cas9) to a target site where it recognizes its PAM sequence (NTT) on the opposition of gRNA-targeted strand. Given that plants such as Arabidopsis have higher genomic AT content, the novel PAM sequence requirement expands targetable genomic sequences, in its own right. Additionally, Cpf1, like ZFN and TALEN, creates staggered DSBs with 50 5 bp overhangs which could be useful for introducing new DNA with compatible sticky ends by the NHEJ pathway. Cpf1 could soon emerge as a powerful and unique alternative to Cas9 for plants, with increased flexibility and efficiency for certain applications. Author contribution statement approved the final manuscript.

Both authors wrote and

Acknowledgments Due to limited space, we could not cite the entirety of the current literature that may also be important. We thank A. Malzahn for critical reading of the manuscript and thoughtful advice on its composition and content. This work is supported by startup funds from East Carolina University and a Collaborative Funding Grant (2016-CFG-8003) from North Carolina Biotechnology Center and Syngenta to YQ. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict interests.

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