review article the power of crispr-cas9-induced genome...

Review ArticleThe Power of CRISPR-Cas9-Induced Genome Editing toSpeed Up Plant Breeding

Hieu X Cao1 Wenqin Wang2 Hien T T Le3 and Giang T H Vu1

1Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Corrensstrasse 3 Gatersleben 06466 Stadt Seeland Germany2School of Agriculture and Biology Shanghai Jiaotong University 800 Dong Chuan Road Shanghai 200240 China3Institute of Genome Research Vietnam Academy of Science and Technology 18 Hoang Quoc Viet Road Cau Giay Hanoi Vietnam

Correspondence should be addressed to Hieu X Cao caoipk-gaterslebende

Received 10 August 2016 Revised 17 October 2016 Accepted 1 November 2016

Academic Editor Graziano Pesole

Copyright copy 2016 Hieu X Cao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Genome editing with engineered nucleases enabling site-directed sequencemodifications bears a great potential for advanced plantbreeding and crop protection Remarkably the RNA-guided endonuclease technology (RGEN) based on the clustered regularlyinterspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy toolthat revolutionizes both basic research and plant breeding Here we review the major technical advances and recent applications oftheCRISPR-Cas9 system formanipulation ofmodel and crop plant genomesWe also discuss the future prospects of this technologyin molecular plant breeding

1 Introduction

Under pressure of rapid population growth climate changeand agricultural pests and diseases the next green evo-lution with new technologies is required to address andprovide novel genetic variations to improve yield quality andresistance against biotic and abiotic stresses in crop plantsDuring the last two decades several crop genomes have beenaltered by introduction of one or more foreign genes of highagronomic values to overcome the limitations of conventionalbreeding techniques Promises as well as critics on suchgenetically modified crops have been discussed intensivelyin other reviews (eg [1ndash3]) and are beyond the scope ofthis paper Alternatively and more powerful genome editingallows precise and predictable changes to bemade to the cropgenetic materials and currently revolutionizes crop breeding(eg [4ndash7])

Genome editing with site-specific nucleases introducesDNAdouble-strand breaks (DSBs) at a target site stimulatingcellular DNA repair mechanisms and subsequently resultingin various types of genome modifications such as targetedmutagenesis gene insertion or gene replacement Thetwo main DSB repair pathways in eukaryotic cells are

nonhomologous end-joining (NHEJ) and homologousrecombination (HR) NHEJ often can cause insertions ordeletions potentially producing a gene knockout Whenrepair templates (regions of homology to the sequencesurrounding the DSB) are available the HR machinery canbe recruited to achieve precise modifications (homology-directed repair HDR) such as gene replacement or geneinsertion (Figure 1) Apparently NHEJ is themost commonlyemployed DSB repair mechanism in many organismsincluding higher plants [8 9]

The latest ground-breaking technology for genome edit-ing is the CRISPR-Cas system which was inspired by thebacterial adaptive immunity against invading bacteriophagesIn August 2012 the groups of Jennifer A Doudna at the Uni-versity of California Berkeley and Emmanuelle Charpentierat the Umea University in Sweden (now at the Max PlanckInstitute of Infection Biology in Berlin) [10] showed for thefirst time that a monomeric DNA endonuclease known asCas9 from Streptococcus pyogenes can be easily programmedto cut double-stranded DNA at a specific genomic sequenceusing complementary base pairing of a single-guide RNA(sgRNA Figure 1) The potential to exploit this simplesystem for genome editing in eukaryotic systems (human

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2016 Article ID 5078796 10 pageshttpdxdoiorg10115520165078796

2 International Journal of Genomics

Cas9 protein

Nucleases

PAM sequence

Target DNA

sgRNA

NHEJ

HDRGene knockout by error-prone repair

Gene insertion with end resection

Gene targeting with precise modifications

NHEJ

HDR

Gene insertion with precise modifications

Donor DNA template

DNA mutations

lowast

Optional with different variants

DSB

(A)

(B)

(C)

(D)

(E)

(F)

(G)

lowast

Figure 1 Overview of CRIPR-Cas9 technology for plant genome editing (A)Themost widely used engineeredCRISPR-Cas9 system in plantsutilizes a plant-codon-optimizedCas9 protein and (could bemore than one) single-guideRNA (sgRNA)Optionally the gene targeting systemwith geminivirus replicons includes an additional donor DNA template (B) In plant cells sgRNA associated with Cas9 nuclease mediatescleavage of target DNA sites that are complementary to the sgRNA and locate next to a PAM sequence (C) Cas9-induced double-strandDNA breaks (DSBs) can be repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways (D) ImpreciseNHEJ-mediated repair can generate insertion andor deletion mutations with variable length at the site of the DSB These InDels can causeout-of-frame mutations in the coding sequences of the target genes resulting in gene knockout (E) In the presence of donor DNA NHEJcan insert the donor DNA into the site of the DSB together with possibly additional InDel mutations HR-driven repair can produce precisemodifications including point mutations (F) or insertions from double-single-strand DNAs as donor templates (G)

mouse) was demonstrated few months later by the work ofFeng Zhangrsquos group at Massachusetts Institute of Technology(MIT) [11] and George Churchrsquos group at Harvard University[12] In these studies only a single construct for expressionof Cas9 nuclease and a specifically designed sgRNA are

needed for transformation Since then due to its ease ofimplementation and robustness the CRISPR-Cas9 systemhas been utilized widely for genome engineering in variousorganisms including plants [13ndash15] insects [16] fish [17] rab-bits [18] pigs [19] mice [20] monkeys [21] and human cells

International Journal of Genomics 3

[22 23] A large number of publications using the CRISPR-Cas9 technology came up rapidly since the first reports andpromoted our understanding and applications of the systemHere we review the major advances in plant genome editingtechnology using Cas9 RNA-guided endonuclease (RGEN)and discuss its applications as well as future prospects inmolecular plant breeding

2 Overview of the Genome EditingCRISPR-Cas9 System

The immense versatility of the CRISPR-Cas9 technology inthe field of genome editing is due to its simplicity efficiencyand robustness Basically the CRISPR-Cas9 tool consistsof two main components deliverable as a single plasmid(Figure 1(A)) a bacterial Cas9 endonuclease protein and aspecifically designed sgRNA containing a 20-bp sequencehomologous to the target DNA (called protospacer) Aprerequisite for cleavage of the target DNA is the presence of asequence 51015840-NGG-31015840 [10] or 51015840-NAG-31015840 [24] as the conservedprotospacer-adjacent motif (PAM) Importantly it has beenshown that multiple sgRNA targeting to different genomicloci can be simultaneously exploited to achieve high-efficiency multiplex genome engineering without requiringadditional Cas9 proteins [11 12] Moreover some initial invitro and in vivo evidence suggested that Cas9 endonucleaseactivity is not affected by DNA CpG methylation [24] How-ever sgRNA preferentially binds to open chromatin regionsincluding off-target sites [25 26] Further efforts to improveour understanding of the effect of chromatin accessibility andepigenetic environment at the target site on the efficiency ofthe CRISPR-Cas9 system are needed

The main concern regarding the implementation of theCRISPR-Cas9 system for genome editing is occasional off-target modifications reported in some studies [11 12 24 27ndash29] Although a 20 bp recognition sequence in the sgRNAwasinitially considered necessary to determine specificity it waslater shown that a perfect match between the 7ndash12 bp at the 31015840end of the sgRNA (called the seed region) and the equivalentregion of the target DNA confers target site recognition andcleavage whereas multiple mismatches in the PAM-distalregion are generally tolerated [24 27ndash29] Several strategieshave been developed to control the specificity of CRISPR-Cas9 in which the design of the sgRNA is considered asan important and easily implementable one A number ofguidelines and online tools have been developed to facilitatethe selection of unique target sites in organisms for whichhigh quality whole genome sequences are available [24 3031] Truncated sgRNA with length of 17 bp or elongatedsgRNA with 2 additional guanidine residues at the 51015840 endcould reduce nontarget mutations [32 33] Low expressionlevel of Cas9 nuclease is another way to reduce off-targetactivities [29 34]

The most widely used Cas9 nuclease originates fromthe type II (class 2) CRISPR-Cas9 system of Streptococ-cus pyogenes (SpCas9) However Cas9 orthologues fromother bacterial species are also applicable and may offerfurther optimization of the current CRIPSR-Cas9 system Forinstance Cas9 gene of Staphylococcus aureus (SaCas9) which

is 1 kb shorter than that of S pyogenes could improve itsstability in transformation vectors [35] Interestingly SaCas9targets another distinct PAM 51015840NNGGGT31015840 On the otherhand a new endonuclease of the class 2 CRISPR-Cas systemsCpf1 (CRISPR from Prevotella and Francisella 1) has beenreported to require a T-rich PAMmotif upstreamof the targetsite and generates a DSB with 51015840 overhangs [36] Finding offurther Cas9 nucleases which require different PAM motifsmight allow targeting of more diverse genomic positions andenable harnessing more complex applications for genomeengineering by using combination of these Cas9

Detailed understanding of the molecular structure of theCRISPR-Cas9 systems guides us to rationally redesign andcustomize variants of Cas9 enzymes Crystal structures ofSpCas9 SaCas9 Francisella novicida Cas9 (FnCas9) or Cpf1in complex with their sgRNA and double-stranded targetDNAs [37ndash41] were solved revealing distinct mechanismsof PAM recognition and of RNA-guided DNA targeting byCas9 nucleases Several engineered CRISPR-Cas9 variantswere produced to increase Cas9 specificity or to alter thePAM recognition patterns [42] Cas9 nickase variants (Cas9-D10A or Cas9-H840A) containing a single inactive nucleasedomain cleave only one DNA strand to create a single-strandbreak at the target sites A pair of induced nicks one on eachstrand and up to 100 bp apart from each other can result in aDSB with overhang This approach using Cas9 nickase couldsignificantly reduce the off-target mutation rate [43 44] Inaddition fusion of catalytically inactive Cas9 (Cas9-D10A-H840A dCas9) and FokI nuclease which functions only asa dimer showed comparable results when they are guidedby a pair of sgRNAs [45 46] Interestingly dCas9 could beexploited not only in genome editing but also in many otherapplications such as modifications of gene expression [47]epigenetic editing [48] and visualization of specific DNAsequences in living cells [49]

3 Major Advances of Plant and Crop GenomeEditing Technology Using Cas9 RNA-GuidedEndonucleases (RGENs)

The CRISPR-Cas9 system with the ability to precisely cutDNA of essentially any organism provides an unprecedentedtool for genomic engineering Soon after the evidence thatthe CRISPR-Cas9 system works in animal models threepapers reported expression and activities of the plant-codon-optimized CRISPR-Cas9 in plant model species of Arabidop-sis (Arabidopsis thaliana) and tobacco (Nicotiana benthami-ana) as well as in crops such as rice and wheat [50ndash52]Thosefirst groups demonstrated the versatility of the technology byusing different transient or stable transformation platforms(protoplast transfection leaf agroinfiltration and particlebombardment of callus) in order to generate small deletionsandor insertions targeted insertions and multiplex genomemodifications Furthermore the transmission to progeny andMendelian heritability of CRISPR-Cas9-induced mutationswas shown by using the Agrobacterium-mediated germ linetransformation in Arabidopsis [14 15 53 54] and rice [55ndash58] suggesting that the CRISPR-Cas9 system could becomea powerful tool in crop genome editing Subsequent work


reported successful applications of the CRISPR-Cas9 toolfor sorghum [13] wheat [59] maize [60] sweet orange [61]tomato [62] potato [63] liverwort Marchantia polymorphaL [64] barley and Brassica oleracea [65] soybean [66]melon [67] and poplar [68] Summarizing information abouttransformationdelivery methods and expression systems forCRISPR-Cas9-based applications in plants can be found inrecent reviews [69 70] It is needed to emphasise that spread-ing of this technology is highly promoted by the CRISPRresearch community providing open access to plasmids webtools and active discussion groups (Table 1)

Since plant genomes are large complex and often poly-ploid off-target mutations can be expected to happen dur-ing genome engineering When introducing a gRNA-Cas9cleavage in rice Xie and Yang [71] reported a mutation rateof 16 at a single off-target sequence which has a singlemismatch at position 15 bp proximal to the PAM Off-targeteffects of CRISPR-Cas9 have been observed in other plantspecies including soybean [72] maize [73] and barley andB oleracea [65] In contrast no off-target mutation eventscould be detected at the putative off-target sites in studieson Arabidopsis tobacco wheat rice or sweet orange [1450ndash52 58 59 61] even using whole-genome sequencing[14] Notably off-target events are less problematic in plantbreeding than for clinical research because off-target muta-tions can be segregated away from the mutation of thetarget by crossing mutants with wild-type plants Howeverthe crossing procedure can be laborious time-consumingor even impossible for perennial plants and vegetativelypropagated crops such as potatoes bananas and cassavaThe off-target problem can be overcome either by optimizingsgRNA design [74 75] or by using high fidelity CRISPR-Cas9 approaches with more precise Cas9 variants (eg [76])paired Cas9 nickases or dCas9FokI fusions The applicationof the Cas9-D10A nickase in Arabidopsis suggests off-targeteffects might be avoided by using a pair of nickases [15 77]

The primary application of CRISPR-Cas9 technologyin genome editing (or reverse genetics studies) is geneknockout because the host cells preferentially repair theCas9-induced DSBs via the ldquoerror-pronerdquo NHEJ pathway whichoften results in short insertions or deletions The size ofthese modifications and the ratio between insertions anddeletions could have an impact on genome size and directgenome size evolution [78 79]The flexibility of the CRISPR-Cas9 tool enables targeting of adjacent sites in chromosomefor specific removal of a large unwanted DNA sequencesfrom several kb in Arabidopsis [52 53] tobacco [30] andtomato [62] up to 245 kb in rice [58] CRISPR-Cas9 canalso be utilized to knockout multiple genes of gene familyin rice [80] or homoeologous genes in hexaploid breadwheat [81 82] A CRISPR-Cas9 toolbox for multiplexedgenome editing was demonstrated in model plants such asArabidopsis tobacco and rice [83] In order to performhighlymultiplexing genome manipulations CRISPR-Cas systemsusing Cas9 orthologues of Staphylococcus aureus (SaCas9)and Streptococcus thermophilus (St1Cas9) have been adaptedfor use in Arabidopsis [84] In addition modified Cas9variants enable targeting to noncanonical PAM sites in rice[85] providing a wider range of genome editing

For site-specific gene insertion (ldquotrait stackingrdquo) orreplacement it is needed to exploit theHR pathway for repair-ing the Cas9-induced DSB HDR events require as templatea sequence homologous to the target gene (Figure 1 [8])However HDR frequency in CRISPR-Cas9-mediated genetargeting (GT) is rather low as shown in rice [50] in tobacco[52] and in Arabidopsis [53 55] By using approximately670 bp homology on either side of the break Schiml et al [77]could insert a 18 kb marker gene into an endogenous gene ofArabidopsis with a frequency of 014 In order to overcomethe low HDR efficiency of targeted genome manipulation inmammalian cells components of the NHEJ pathway wereinhibited [86] Similarly by manipulating of DNA ligase IV(a member of the NHEJ pathway) CRISPR-Cas9-inducedHDR-mediated GT can work more efficiently in rice result-ing in biallelic plants [87] Alternatively rational design oforientation polarity and length of the donor ssDNA tomatchthe properties of the Cas9-DNA complex could increase theHDR events [88] Ideally the efficiency of HDR-mediatedgenome modifications would be improved by delivery ofsufficient quantities of the donor sequence for HDR repairat the Cas9-targeted site A transformation method using anuclear replicating DNA virus [89] which produces multiplecopies of the donor sequences for HDR inside plant cellshas recently been demonstrated to generate high-frequencyprecise genome modifications in tomato [90]

4 Future Perspectives of the CRISPR-CASTechnology for Plant Breeding

The CRISPR-Cas9 technology is revolutionizing genomeengineering and equipping scientists and breeders with theability to precisely modify the DNA of crop plants Impor-tantly CRISPR-Cas9 enables genome modifications also inpotential crop plants forwhich geneticmanipulation has beena challenge (eg duckweed [91]) provided that high qualitywhole genome sequences [92 93] and efficient transforma-tion procedures are available [94]This review does not coverethical legal and social issues of this revolutionary tool (forsuch aspects see [2ndash4 95]) In this context it is necessaryto note that the common white button mushroom (Agaricusbisporus) that has been modified to resist browning usingCRISPR-Cas9 became the first CRISPR-edited organism thatcan be cultivated and sold without further oversight of USregulations [96] Interestingly the CRISPR-Cas9 approachoffers genetic manipulation of crops without transgenicfootprints by delivering preassembled Cas9-sgRNA ribonu-cleoproteins [97] or by transient expression of the in vitrotranscripts of Cas9-coding sequence and sgRNA [82] andthusmight not be classified as geneticallymodified organismsand regulated by existing biosafety regulations

Amajor power of CRISPRCas9-induced genome editingis to provide an opportunity for targetingmultiple sites simul-taneously New application of this technology is conferringmultiple pathogen resistances to crop plants By establishingCRISPR-Cas9-like immune systems tobacco andArabidopsisweremade resistant to the beet severe curly top virus [98] thebean yellow dwarf virus [99] and the tomato yellow leaf curlvirus respectively [100]The recently developed CRISPR-Cas


Table1Usefulresou

rcesto

olsfor

CRISPR

-Cas9research

inplants

Site

Purpose

Authority

httpwwwaddgeneo

rgAc

cessto

plasmid

resource

andtutoria

ldocum

ents[115]

Addgene

httpsw

wwprotocolsio

Accessto

detailedprotocolresource

Protocolsio

httpcbih

zaueducncgi-binCRISP

RDesignop

timalsgRN

Awith

43plantgenom

esfro

mEn

semblPlants[24]

Huazhon

gAgricultural

University

httpwwwgeno

mea

rizon

aedu

cris

prCRISP

Rsearchhtml

Predicth

ighspecifics

gRNAof

8plantgenom

es[116]

University

ofAriz

ona

httpwwwrgenom

enetcas-offind

er

Search

forp

otentia

loff-targetsites

ofsgRN

Afro

m37

plantgenom

es[117]

InstituteforB

asic

ScienceKo

rea

httpwwwe-crisp

orgE-C

RISP

indexhtml

DesignsgRN

Aforg

enom

e-lib

rarie

sprojectso

rindividu

alsequ

encesw

ith11plantgenom

es[118]

German

Cancer

Research

Center(DKF

Z)

httpcrisp

rmitedu

Find

theC

RISP

R-Ca

s9targetsites

with

inan

inpu

tsequ

ence

with

Arabidopsis

geno

me[24]

ZhangLabMIT

httpchop

chopcb

uuibno

SelectCR

ISPR

targetsites

andpredicto

ff-targetsites

with

Arabidopsis

geno

me[119

]University

ofBe

rgen

httppo

rtalsb

roadinstituteorggpp

pub

licanalysis

-toolssgrna-desig

nDesignhigh

lyactiv

esgR

NAs

forthe

provided

targets

[74]

BroadInstituteof

MIT

andHarvardC

ambridge

University

httpeend

bzfgenetic

sorgcasot

Open-sourcedtoolforfi

ndingpo

tentialoff-targetsites

ofanyuser-specific

geno

me[120]

Peking

University

httpsgroup

sgoo

glec

omfo

rumfo

rumcris

prAc

tived

iscussio

ngrou

psGoo

gle


system with programmable RNA recognition and cleavage[101] would be exciting to apply in plants because themajorityof plant viruses are RNA viruses [102] However furtherstudies will be required to monitor the stability of suchresistances over generations and in diverse habitats [103]

CRISPR-Cas9 has triggered innovative applications inseveral fields including agriculture CRISPR-edited indi-vidual organisms could spread a positively selectable genethroughout a wild population in a so-called gene driveprocess In principle such CRISPR-based gene drive systemscould be beneficial to mankind for example by potentiallypreventing the spread of diseases or supporting agricultureby reversing pesticide and herbicide resistance in insectsand weeds and control damaging invasive species [104]Gene drives will work only in sexually reproducing speciesand spread significantly only in species that reproducequickly The gene drive model has been tested in yeast[105] and the first CRISPR-Cas9-engineered mosquitoeshave recently developed to fight malaria [106] Howeverbecause of low efficient homologous recombination genedrive application to either eliminate or reduce invasive plantspecies in a given area is still challenging In addition sincesuch technology might pose tremendous alterations to wildpopulations biosafety precautions and measures are needed(for more details see [107 108]) Importantly the CRISPR-Cas9-mediated gene drive technology (called as mutagenicchain reaction (MCR) [109]) can be used to produce stablehomozygous (biallelic) mutant lines by using HDR-drivenpropagation of the CRISPR-Cas9-cassette to the companionchromosome Moreover such concept can also be appliedfor editing organelle genomes (eg chloroplast) in order toovercome the high copy number of genomes and reversion ofmutations [110]

Until now synthetic biology is limited to bacteria mod-els to engineer completely new metabolic pathways TheCRISPR-Cas9 technology opens the way to an easier use ofsynthetic biology in more complex systems for example foragronomical traits in crop plants [111] Since many complexmetabolic pathways in plants interact with each other and arecontrolled by multiple tissue- or development-specific regu-lators metabolic engineering in plants could require not onlymultiple gene targeting but also probably fine-tuningmultiplegene expression level at different tissues or developmentalstages For such sophisticated applications modifications orcustomizations of the CRISPR-Cas9 systems including (1)specific Cas9-sgRNA expression promoters (eg [112 113])(2) modified Cas9 for alterations of gene expression and epi-genetic changes (eg [47 48]) (3) combinations of differentCas9 variants (eg [35 36]) for expanding the target rangein the genomes and (4) efficient technology for increasingHDR-driven precise gene replacement will be needed to befurther developed or optimized for particular cell types ororganisms With the rapid development of CRISPR-Cas9technology during the last 4 years the promise of a next greenrevolution with new crops meeting long-standing requestsfor metabolic engineering (eg plants that can fix their ownnitrogen have better nutritious values can be efficientlyutilized for biofuel production or display enhanced photo-synthetic capacity [114]) could be realized in the near future

Abbreviations

CRISPR Clustered regularly interspaced shortpalindromic repeats

Cas9 CRISPR-associated protein 9DSB DNA double-strand breakGT Gene targetingHR Homologous recombinationNHEJ Nonhomologous end-joiningsgRNA Single-guide RNAPAM Protospacer-adjacent motifRGEN RNA-guided endonuclease

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Ingo Schubert from the Leibniz Instituteof Plant Genetics and Crop Plant Research (IPK) for criticalreading of the manuscript

References

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[2] R R Aldemita I M Reano R O Solis and R A HautealdquoTrends in global approvals of biotech crops (1992ndash2014)rdquo GMCrops amp Food vol 6 no 3 pp 150ndash166 2015

[3] M Araki and T Ishii ldquoTowards social acceptance of plantbreeding by genome editingrdquo Trends in Plant Science vol 20no 3 pp 145ndash149 2015

[4] S Huang D Weigel R N Beachy and J Li ldquoA proposed reg-ulatory framework for genome-edited cropsrdquo Nature Geneticsvol 48 no 2 pp 109ndash111 2016

[5] S Schiml and H Puchta ldquoRevolutionizing plant biologymultiple ways of genome engineering by CRISPRCasrdquo PlantMethods vol 12 article 8 2016

[6] K Belhaj A Chaparro-Garcia S Kamoun N J Patron and VNekrasov ldquoEditing plant genomes with CRISPRCas9rdquo CurrentOpinion in Biotechnology vol 32 pp 76ndash84 2015

[7] D Carroll ldquoGenome engineering with targetable nucleasesrdquoAnnual Review of Biochemistry vol 83 pp 409ndash439 2014

[8] G T H Vu H X Cao K Watanabe et al ldquoRepair of site-specific DNA double-strand breaks in barley occurs via diversepathways primarily involving the sister chromatidrdquo Plant Cellvol 26 no 5 pp 2156ndash2167 2014

[9] H Puchta ldquoThe repair of double-strand breaks in plantsmechanisms and consequences for genome evolutionrdquo Journalof Experimental Botany vol 56 no 409 pp 1ndash14 2005

[10] M Jinek K Chylinski I Fonfara M Hauer J A Doudnaand E Charpentier ldquoA programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunityrdquo Science vol 337no 6096 pp 816ndash821 2012

[11] L Cong F A RanD Cox et al ldquoMultiplex genome engineeringusing CRISPRCas systemsrdquo Science vol 339 no 6121 pp 819ndash823 2013


[12] P Mali L Yang K M Esvelt et al ldquoRNA-guided humangenome engineering via Cas9rdquo Science vol 339 no 6121 pp823ndash826 2013

[13] W Jiang H Zhou H Bi M Fromm B Yang and D P WeeksldquoDemonstration of CRISPRCas9sgRNA-mediated targetedgene modification in Arabidopsis tobacco sorghum and ricerdquoNucleic Acids Research vol 41 no 20 article e188 2013

[14] Z Feng Y Mao N Xu et al ldquoMultigeneration analysisreveals the inheritance specificity and patterns of CRISPRCas-induced gene modifications in Arabidopsisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 111 no 12 pp 4632ndash4637 2014

[15] F Fauser S Schiml and H Puchta ldquoBoth CRISPRCas-basednucleases and nickases can be used efficiently for genomeengineering in Arabidopsis thalianardquo Plant Journal vol 79 no2 pp 348ndash359 2014

[16] A R Bassett C Tibbit C P Ponting and J-L Liu ldquoHighlyEfficient TargetedMutagenesis of Drosophila with the CRISPRCas9 Systemrdquo Cell Reports vol 4 no 1 pp 220ndash228 2013

[17] W Y Hwang Y Fu D Reyon et al ldquoEfficient genome editingin zebrafish using a CRISPR-Cas systemrdquoNature Biotechnologyvol 31 no 3 pp 227ndash229 2013

[18] A Honda M Hirose T Sankai et al ldquoSingle-step generationof rabbits carrying a targeted allele of the tyrosinase gene usingCRISPRCas9rdquo Experimental Animals vol 64 no 1 pp 31ndash372015

[19] K M Whitworth K Lee J A Benne et al ldquoUse of theCRISPRCas9 system to produce genetically engineered pigsfrom in vitro-derived oocytes and embryosrdquo Biology of Repro-duction vol 91 no 3 article no 78 2014

[20] D Seruggia A Fernandez M Cantero P Pelczar and LMontoliu ldquoFunctional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediatedmutagenesisrdquo Nucleic Acids Research vol 43 no 10 pp 4855ndash4867 2015

[21] Y Niu B Shen Y Cui et al ldquoGeneration of gene-modifiedcynomolgus monkey via Cas9RNA-mediated gene targeting inone-cell embryosrdquo Cell vol 156 no 4 pp 836ndash843 2014

[22] S W Cho S Kim J M Kim and J-S Kim ldquoTargetedgenome engineering in human cells with the Cas9 RNA-guidedendonucleaserdquoNature Biotechnology vol 31 no 3 pp 230ndash2322013

[23] M Jinek A East A Cheng S Lin EMa and J Doudna ldquoRNA-programmed genome editing in human cellsrdquo Elife vol 2013no 2 Article ID e00471 2013

[24] P D Hsu D A Scott J A Weinstein et al ldquoDNA targetingspecificity of RNA-guided Cas9 nucleasesrdquo Nature Biotechnol-ogy vol 31 no 9 pp 827ndash832 2013

[25] X Wu D A Scott A J Kriz et al ldquoGenome-wide binding ofthe CRISPR endonuclease Cas9 in mammalian cellsrdquo NatureBiotechnology vol 32 no 7 pp 670ndash676 2014

[26] C Kuscu S Arslan R Singh JThorpe andM Adli ldquoGenome-wide analysis reveals characteristics of off-target sites bound bythe Cas9 endonucleaserdquo Nature Biotechnology vol 32 no 7 pp677ndash683 2014

[27] Y Fu J A Foden C Khayter et al ldquoHigh-frequency off-targetmutagenesis induced by CRISPR-Cas nucleases in human cellsrdquoNature Biotechnology vol 31 no 9 pp 822ndash826 2013

[28] W Y Jiang D Bikard D Cox F Zhang and L A MarraffinildquoRNA-guided editing of bacterial genomes using CRISPR-Cassystemsrdquo Nature Biotechnology vol 31 no 3 pp 233ndash239 2013

[29] V Pattanayak S Lin J P Guilinger E Ma J A Doudna and DR Liu ldquoHigh-throughput profiling of off-target DNA cleavagereveals RNA-programmed Cas9 nuclease specificityrdquo NatureBiotechnology vol 31 no 9 pp 839ndash843 2013

[30] K Belhaj A Chaparro-Garcia S Kamoun and V NekrasovldquoPlant genome editing made easy targeted mutagenesis inmodel and crop plants using the CRISPRCas systemrdquo PlantMethods vol 9 article 39 2013

[31] J G Doench E Hartenian D B Graham et al ldquoRationaldesign of highly active sgRNAs for CRISPR-Cas9-mediatedgene inactivationrdquo Nature Biotechnology vol 32 no 12 pp1262ndash1267 2014

[32] Y F Fu J D Sander D Reyon V M Cascio and J K JoungldquoImproving CRISPR-Cas nuclease specificity using truncatedguide RNAsrdquo Nature Biotechnology vol 32 no 3 pp 279ndash2842014

[33] S W Cho S Kim Y Kim et al ldquoAnalysis of off-targeteffects of CRISPRCas-derived RNA-guided endonucleases andnickasesrdquo Genome Research vol 24 no 1 pp 132ndash141 2014

[34] W Fujii K Kawasaki K Sugiura and K Naito ldquoEfficientgeneration of large-scale genome-modified mice using gRNAand CAS9 endonucleaserdquoNucleic Acids Research vol 41 no 20article e187 2013

[35] F A Ran L Cong W X Yan et al ldquoIn vivo genome editingusing Staphylococcus aureus Cas9rdquo Nature vol 520 no 7546pp 186ndash191 2015

[36] B Zetsche J S Gootenberg O O Abudayyeh et al ldquoCpf1 Isa single RNA-guided endonuclease of a class 2 CRISPR-Cassystemrdquo Cell vol 163 no 3 pp 759ndash771 2015

[37] H Nishimasu F A Ran P D Hsu et al ldquoCrystal structure ofCas9 in complex with guide RNA and target DNArdquo Cell vol156 no 5 pp 935ndash949 2014

[38] H Nishimasu L Cong W X Yan et al ldquoCrystal Structure ofStaphylococcus aureus Cas9rdquo Cell vol 162 no 5 pp 1113ndash11262015

[39] C Anders O Niewoehner A Duerst andM Jinek ldquoStructuralbasis of PAM-dependent target DNA recognition by the Cas9endonucleaserdquo Nature vol 513 no 7519 pp 569ndash573 2014

[40] T Yamano H Nishimasu B Zetsche et al ldquoCrystal structureof Cpf1 in complex with guide RNA and target DNArdquo Cell vol165 no 4 pp 949ndash962 2016

[41] H Hirano J Gootenberg T Horii et al ldquoStructure andEngineering of Francisella novicida Cas9rdquo Cell vol 164 no 5pp 950ndash961 2016

[42] S Hirano H Nishimasu R Ishitani and O Nureki ldquoStructuralbasis for the altered PAM specificities of engineered CRISPR-Cas9rdquoMolecular Cell vol 61 no 6 pp 886ndash894 2016

[43] F A Ran P D Hsu C-Y Lin et al ldquoDouble nicking by RNA-guided CRISPR cas9 for enhanced genome editing specificityrdquoCell vol 154 no 6 pp 1380ndash1389 2013

[44] B Shen W Zhang J Zhang et al ldquoEfficient genome modifica-tion by CRISPR-Cas9 nickase with minimal off-target effectsrdquoNature Methods vol 11 no 4 pp 399ndash402 2014

[45] J P Guilinger D B Thompson and D R Liu ldquoFusionof catalytically inactive Cas9 to FokI nuclease improves thespecificity of genome modificationrdquo Nature Biotechnology vol32 no 6 pp 577ndash582 2014

[46] S Q Tsai N Wyvekens C Khayter et al ldquoDimeric CRISPRRNA-guided FokI nucleases for highly specific genome editingrdquoNature Biotechnology vol 32 no 6 pp 569ndash576 2014


[47] L S QiMH Larson L A Gilbert et al ldquoRepurposingCRISPRas an RNA-guided platform for sequence-specific control ofgene expressionrdquo Cell vol 152 no 5 pp 1173ndash1183 2013

[48] I B Hilton A M DrsquoIppolito C M Vockley et al ldquoEpigenomeediting by a CRISPR-Cas9-based acetyltransferase activatesgenes from promoters and enhancersrdquo Nature Biotechnologyvol 33 no 5 pp 510ndash517 2015

[49] B Chen L A Gilbert B A Cimini et al ldquoDynamic imaging ofgenomic loci in living human cells by an optimizedCRISPRCassystemrdquo Cell vol 155 no 7 pp 1479ndash1491 2013

[50] Q Shan Y Wang J Li et al ldquoTargeted genome modification ofcrop plants using a CRISPR-Cas systemrdquo Nature Biotechnologyvol 31 no 8 pp 686ndash688 2013

[51] V Nekrasov B Staskawicz D Weigel J D G Jones and SKamoun ldquoTargeted mutagenesis in the model plant Nicotianabenthamiana using Cas9 RNA-guided endonucleaserdquo NatureBiotechnology vol 31 no 8 pp 691ndash693 2013

[52] J-F Li J E Norville J Aach et al ldquoMultiplex and homologousrecombination-mediated genome editing in ArabidopsisandNicotiana benthamianausing guide RNA and Cas9rdquo NatureBiotechnology vol 31 no 8 pp 688ndash691 2013

[53] Y Mao H Zhang N Xu B Zhang F Gou and J-K ZhuldquoApplication of the CRISPR-Cas system for efficient genomeengineering in plantsrdquoMolecular Plant vol 6 no 6 pp 2008ndash2011 2013

[54] W Jiang B Yang and D P Weeks ldquoEfficient CRISPRCas9-mediated gene editing in Arabidopsis thaliana and inheritanceofmodified genes in the T2 and T3 generationsrdquo PLoSONE vol9 no 6 Article ID e99225 2014

[55] Z Feng B Zhang W Ding et al ldquoEfficient genome editing inplants using a CRISPRCas systemrdquo Cell Research vol 23 no10 pp 1229ndash1232 2013

[56] H Zhang J Zhang P Wei et al ldquoThe CRISPRCas9 systemproduces specific and homozygous targeted gene editing in ricein one generationrdquo Plant Biotechnology Journal vol 12 no 6pp 797ndash807 2014

[57] J Miao D S Guo J Z Zhang et al ldquoTargeted mutagenesis inrice using CRISPR-Cas systemrdquoCell Research vol 23 no 10 pp1233ndash1236 2013

[58] H Zhou B Liu D P Weeks M H Spalding and B YangldquoLarge chromosomal deletions and heritable small geneticchanges induced by CRISPRCas9 in ricerdquo Nucleic AcidsResearch vol 42 no 17 pp 10903ndash10914 2014

[59] S K Upadhyay J Kumar A Alok and R Tuli ldquoRNA-guidedgenome editing for target gene mutations in wheatrdquo G3 GenesGenomes Genetics vol 3 no 12 pp 2233ndash2238 2013

[60] Z Liang K Zhang K Chen andCGao ldquoTargetedmutagenesisin Zea mays using TALENs and the CRISPRCas systemrdquoJournal of Genetics and Genomics vol 41 no 2 pp 63ndash68 2014

[61] H Jia and N Wang ldquoTargeted genome editing of sweet orangeusing Cas9sgRNArdquo PLoS ONE vol 9 no 4 Article ID e938062014

[62] C Brooks V Nekrasov Z B Lippman and J Van Eck ldquoEfficientgene editing in tomato in the first generation using the clus-tered regularly interspaced short palindromic repeatsCRISPR-associated9 systemrdquo Plant Physiology vol 166 no 3 pp 1292ndash1297 2014

[63] N M Butler P A Atkins D F Voytas and D S DouchesldquoGeneration and inheritance of targeted mutations in potato(Solanum tuberosum L) using the CRISPRCas Systemrdquo PLoSONE vol 10 no 12 Article ID e0144591 2015

[64] S S Sugano M Shirakawa J Takagi et al ldquoCRISPRCas9-mediated targeted mutagenesis in the liverwort Marchantiapolymorpha Lrdquo Plant and Cell Physiology vol 55 no 3 pp 475ndash481 2014

[65] T Lawrenson O Shorinola N Stacey et al ldquoInduction oftargeted heritable mutations in barley and Brassica oleraceausing RNA-guided Cas9 nucleaserdquo Genome Biology vol 16article 258 2015

[66] T B Jacobs P R LaFayette R J Schmitz and W A ParrottldquoTargeted genome modifications in soybean with CRISPRCas9rdquo BMC Biotechnology pp 1ndash10 2015

[67] G Tzuri X Zhou N Chayut et al ldquoA lsquogoldenrsquo SNP in CmOrgoverns the fruit flesh color of melon (Cucumis melo)rdquo PlantJournal vol 82 no 2 pp 267ndash279 2015

[68] D Fan T Liu C Li et al ldquoEfficient CRISPRCas9-mediated tar-geted mutagenesis in populus in the first generationrdquo ScientificReports vol 5 Article ID 12217 2015

[69] X Ma Q Zhu Y Chen and Y G Liu ldquoCRISPRCas9 platformsfor genome editing in plants developments and applicationsrdquoMolecular Plant vol 9 no 7 pp 961ndash974 2016

[70] S M Schaeffer and P A Nakata ldquoThe expanding footprint ofCRISPRCas9 in the plant sciencesrdquo Plant Cell Reports vol 35no 7 pp 1451ndash1468 2016

[71] K Xie and Y Yang ldquoRNA-guided genome editing in plantsusing a CRISPR-Cas systemrdquo Molecular Plant vol 6 no 6 pp1975ndash1983 2013

[72] X Sun Z Hu R Chen et al ldquoTargeted mutagenesis in soybeanusing the CRISPR-Cas9 systemrdquo Scientific Reports vol 5 article10342 2015

[73] C Feng J Yuan R Wang Y Liu J A Birchler and FHan ldquoEfficient targeted genome modification in maize usingCRISPRCas9 systemrdquo Journal of Genetics and Genomics vol43 no 1 pp 37ndash43 2016

[74] J G Doench N Fusi M Sullender et al ldquoOptimized sgRNAdesign to maximize activity and minimize off-target effects ofCRISPR-Cas9rdquoNature Biotechnology vol 34 no 2 pp 184ndash1912016

[75] S Q Tsai and J K Joung ldquoDefining and improving the genome-wide specificities of CRISPR-Cas9 nucleasesrdquo Nature ReviewsGenetics vol 17 no 5 pp 300ndash312 2016

[76] B P Kleinstiver V Pattanayak M S Prew et al ldquoHigh-fidelityCRISPR-Cas9 nucleases with no detectable genome-wide off-target effectsrdquo Nature vol 529 no 7587 pp 490ndash495 2016

[77] S Schiml F Fauser and H Puchta ldquoThe CRISPRCas systemcan be used as nuclease for in planta gene targeting and as pairednickases for directed mutagenesis in Arabidopsis resulting inheritable progenyrdquo Plant Journal vol 80 no 6 pp 1139ndash11502014

[78] G T H Vu T Schmutzer F Bull et al ldquoComparative genomeanalysis reveals divergent genome size evolution in a carnivo-rous plant genusrdquo Plant Genome vol 8 no 3 2015

[79] I Schubert and G T Vu ldquoGenome stability and evolutionattempting a holistic viewrdquo Trends in Plant Science vol 21 no9 pp 749ndash757 2016

[80] M Endo M Mikami and S Toki ldquoMultigene knockoututilizing off-target mutations of the CRISPRcas9 system inricerdquo Plant and Cell Physiology vol 56 no 1 pp 41ndash47 2015

[81] Y Wang X Cheng Q Shan et al ldquoSimultaneous editing ofthree homoeoalleles in hexaploid bread wheat confers heritableresistance to powdery mildewrdquo Nature Biotechnology vol 32no 9 pp 947ndash951 2014


[82] Y Zhang Z Liang Y Zong et al ldquoEfficient and transgene-free genome editing in wheat through transient expression ofCRISPRCas9 DNA or RNArdquo Nature Communications vol 7Article ID 12617 2016

[83] L G Lowder D Zhang N J Baltes et al ldquoA CRISPRCas9 tool-box for multiplexed plant genome editing and transcriptionalregulationrdquo Plant Physiology vol 169 no 2 pp 971ndash985 2015

[84] J Steinert S Schiml F Fauser and H Puchta ldquoHighly efficientheritable plant genome engineering using Cas9 orthologuesfrom Streptococcus thermophilus and Staphylococcus aureurdquoPlant Journal vol 84 no 6 pp 1295ndash1305 2015

[85] X Hu CWang Y Fu Q Liu X Jiao and KWang ldquoExpandingthe range of CRISPRCas9 genome editing in ricerdquo MolecularPlant vol 9 no 6 pp 943ndash945 2016

[86] T Maruyama S K Dougan M C Truttmann A M BilateJ R Ingram and H L Ploegh ldquoIncreasing the efficiency ofprecise genome editing with CRISPR-Cas9 by inhibition ofnonhomologous end joiningrdquoNature Biotechnology vol 33 no5 pp 538ndash542 2015

[87] M Endo M Mikami and S Toki ldquoBiallelic gene targeting inricerdquo Plant Physiology vol 170 no 2 pp 667ndash677 2016

[88] C D Richardson G J Ray M A DeWitt G L Curie andJ E Corn ldquoEnhancing homology-directed genome editing bycatalytically active and inactiveCRISPR-Cas9 using asymmetricdonor DNArdquo Nature Biotechnology vol 34 no 3 pp 339ndash3442016

[89] N J Baltes J Gil-Humanes T Cermak P A Atkins and D FVoytas ldquoDNA replicons for plant genome engineeringrdquo PlantCell vol 26 no 1 pp 151ndash163 2014

[90] T Cermak N J Baltes R Cegan Y Zhang and D F VoytasldquoHigh-frequency precise modification of the tomato genomerdquoGenome Biology vol 16 article 232 2015

[91] K-J Appenroth D J Crawford and D H Les ldquoAfterthe genome sequencing of duckweedmdashhow to proceed withresearch on the fastest growing angiospermrdquo Plant Biology vol17 supplement 1 pp 1ndash4 2015

[92] H X Cao G T H Vu W Wang K J Appenroth J Messingand I Schubert ldquoThemap-based genome sequence of Spirodelapolyrhiza aligned with its chromosomes a reference for kary-otype evolutionrdquo New Phytologist vol 209 no 1 pp 354ndash3632016

[93] W Wang G Haberer H Gundlach et al ldquoThe Spirodelapolyrhiza genome reveals insights into its neotenous reductionfast growth and aquatic lifestylerdquo Nature Communications vol5 article 3311 2014

[94] A Canto-Pastor A Molla-Morales E Ernst et al ldquoEfficienttransformation and artificial miRNA gene silencing in Lemnaminorrdquo Plant Biology vol 17 no 1 pp 59ndash65 2015

[95] J D Wolt K Wang and B Yang ldquoThe regulatory status ofgenome-edited cropsrdquo Plant Biotechnology Journal vol 14 no2 pp 510ndash518 2016

[96] E Waltz ldquoGene-edited CRISPR mushroom escapes US regula-tionrdquo Nature vol 532 no 7599 pp 293ndash293 2016

[97] J W Woo J Kim S I Kwon et al ldquoDNA-free genome editingin plants with preassembled CRISPR-Cas9 ribonucleoproteinsrdquoNature Biotechnology vol 33 no 11 pp 1162ndash1164 2015

[98] X Ji H Zhang Y Zhang Y Wang and C Gao ldquoEstablishinga CRISPR-Cas-like immune system conferring DNA virusresistance in plantsrdquoNature Plants vol 1 Article ID 15144 2015

[99] N J Baltes A W Hummel E Konecna et al ldquoConferringresistance to geminiviruses with the CRISPR-Cas prokaryotic

immune systemrdquo Nature Plants vol 1 no 10 Article ID 151452015

[100] Z Ali A Abulfaraj A Idris S Ali M Tashkandi and M MMahfouz ldquoCRISPRCas9-mediated viral interference in plantsrdquoGenome Biology vol 16 no 1 article 238 2015

[101] M R OrsquoConnell B L Oakes S H Sternberg A East-SeletskyM Kaplan and J A Doudna ldquoProgrammable RNA recognitionand cleavage by CRISPRCas9rdquo Nature vol 516 no 7530 pp263ndash266 2014

[102] DD Zhang Z X Li and J F Li ldquoGenome editing new antiviralweapon for plantsrdquoNature Plants vol 1 no 10 Article ID 151462015

[103] A Chaparro-Garcia S Kamoun and V Nekrasov ldquoBoostingplant immunity with CRISPRCasrdquo Genome Biology vol 16article 254 2015

[104] K M Esvelt A L Smidler F Catteruccia and G M ChurchldquoConcerning RNA-guided gene drives for the alteration of wildpopulationsrdquo eLife vol 3 Article ID e03401 2014

[105] J E DiCarlo A Chavez S L Dietz K M Esvelt and GM Church ldquoSafeguarding CRISPR-Cas9 gene drives in yeastrdquoNature Biotechnology vol 33 no 12 pp 1250ndash1255 2015

[106] A Hammond R Galizi K Kyrou et al ldquoA CRISPR-Cas9gene drive system targeting female reproduction in the malariamosquito vector Anopheles gambiaerdquoNature Biotechnology vol34 no 1 pp 78ndash83 2016

[107] O S Akbari H J Bellen E Bier et al ldquoSafeguarding gene driveexperiments in the laboratoryrdquo Science vol 349 no 6251 pp927ndash929 2015

[108] J Champer A Buchman andO S Akbari ldquoCheating evolutionengineering gene drives to manipulate the fate of wild popula-tionsrdquo Nature Reviews Genetics vol 17 no 3 pp 146ndash159 2016

[109] V M Gantz N Jasinskiene O Tatarenkova et al ldquoHighlyefficient Cas9-mediated gene drive for population modificationof the malaria vector mosquito Anopheles stephensirdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 112 no 49 pp E6736ndashE6743 2015

[110] E Martin Avila M F Gisby and A Day ldquoSeamless editing ofthe chloroplast genome in plantsrdquo BMC Plant Biology vol 16no 1 article 168 2016

[111] G Farre R M Twyman P Christou T Capell and CZhu ldquoKnowledge-driven approaches for engineering complexmetabolic pathways in plantsrdquo Current Opinion in Biotechnol-ogy vol 32 pp 54ndash60 2015

[112] Y Hyun J Kim S W Cho Y Choi J-S Kim and GCoupland ldquoSite-directed mutagenesis in Arabidopsis thalianausing dividing tissue-targeted RGENof theCRISPRCas systemto generate heritable null allelesrdquo Planta vol 241 no 1 pp 271ndash284 2015

[113] Z-P Wang H-L Xing L Dong et al ldquoEgg cell-specificpromoter-controlled CRISPRCas9 efficiently generateshomozygous mutants for multiple target genes in Arabidopsisin a single generationrdquo Genome Biology vol 16 article 1442015

[114] W Lau M A Fischbach A Osbourn and E S Sattely ldquoKeyapplications of plant metabolic engineeringrdquo PLoS biology vol12 no 6 Article ID e1001879 2014

[115] J Kamens ldquoTheAddgene repository an international nonprofitplasmid and data resourcerdquo Nucleic Acids Research vol 43 no1 pp D1152ndashD1157 2015

[116] K Xie J Zhang and Y Yang ldquoGenome-wide prediction ofhighly specific guide RNA spacers for CRISPR-Cas9-mediated


genome editing in model plants and major cropsrdquo MolecularPlant vol 7 no 5 pp 923ndash926 2014

[117] S Bae J Park and J-S Kim ldquoCas-OFFinder a fast and versatilealgorithm that searches for potential off-target sites of Cas9RNA-guided endonucleasesrdquo Bioinformatics vol 30 no 10 pp1473ndash1475 2014

[118] F Heigwer G Kerr and M Boutros ldquoE-CRISP fast CRISPRtarget site identificationrdquoNature Methods vol 11 no 2 pp 122ndash123 2014

[119] K Labun T G Montague J A Gagnon S B Thyme and EValen ldquoCHOPCHOP v2 a web tool for the next generation ofCRISPR genome engineeringrdquo Nucleic Acids Research vol 44no W1 pp W272ndashW276 2016

[120] A Xiao Z Cheng L Kong et al ldquoCasOT a genome-wideCas9gRNA off-target searching toolrdquo Bioinformatics vol 30no 8 pp 1180ndash1182 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Cas9 protein

Nucleases

PAM sequence

Target DNA

sgRNA

NHEJ

HDRGene knockout by error-prone repair

Gene insertion with end resection

Gene targeting with precise modifications

NHEJ

HDR

Gene insertion with precise modifications

Donor DNA template

DNA mutations

lowast

Optional with different variants

DSB

(A)

(B)

(C)

(D)

(E)

(F)

(G)

lowast

Figure 1 Overview of CRIPR-Cas9 technology for plant genome editing (A)Themost widely used engineeredCRISPR-Cas9 system in plantsutilizes a plant-codon-optimizedCas9 protein and (could bemore than one) single-guideRNA (sgRNA)Optionally the gene targeting systemwith geminivirus replicons includes an additional donor DNA template (B) In plant cells sgRNA associated with Cas9 nuclease mediatescleavage of target DNA sites that are complementary to the sgRNA and locate next to a PAM sequence (C) Cas9-induced double-strandDNA breaks (DSBs) can be repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways (D) ImpreciseNHEJ-mediated repair can generate insertion andor deletion mutations with variable length at the site of the DSB These InDels can causeout-of-frame mutations in the coding sequences of the target genes resulting in gene knockout (E) In the presence of donor DNA NHEJcan insert the donor DNA into the site of the DSB together with possibly additional InDel mutations HR-driven repair can produce precisemodifications including point mutations (F) or insertions from double-single-strand DNAs as donor templates (G)

mouse) was demonstrated few months later by the work ofFeng Zhangrsquos group at Massachusetts Institute of Technology(MIT) [11] and George Churchrsquos group at Harvard University[12] In these studies only a single construct for expressionof Cas9 nuclease and a specifically designed sgRNA are

needed for transformation Since then due to its ease ofimplementation and robustness the CRISPR-Cas9 systemhas been utilized widely for genome engineering in variousorganisms including plants [13ndash15] insects [16] fish [17] rab-bits [18] pigs [19] mice [20] monkeys [21] and human cells




















Table1Usefulresou

rcesto

olsfor

CRISPR

-Cas9research

inplants

Site

Purpose

Authority

httpwwwaddgeneo

rgAc

cessto

plasmid

resource

andtutoria

ldocum

ents[115]

Addgene

httpsw

wwprotocolsio

Accessto


Protocolsio

httpcbih


RDesignop

timalsgRN

Awith

43plantgenom

esfro

mEn

semblPlants[24]

Huazhon

gAgricultural

University

httpwwwgeno

mea

rizon

aedu

cris

prCRISP

Rsearchhtml

Predicth

ighspecifics

gRNAof

8plantgenom

es[116]

University

ofAriz

ona

httpwwwrgenom

enetcas-offind

er

Search

forp

otentia

loff-targetsites

ofsgRN

Afro

m37

plantgenom

es[117]

InstituteforB

asic

ScienceKo

rea

httpwwwe-crisp

orgE-C

RISP

indexhtml

DesignsgRN

Aforg

enom

e-lib

rarie

sprojectso

rindividu

alsequ

encesw

ith11plantgenom

es[118]

German

Cancer

Research

Center(DKF

Z)

httpcrisp

rmitedu

Find

theC

RISP

R-Ca

s9targetsites

with

inan

inpu

tsequ

ence

with

Arabidopsis

geno

me[24]

ZhangLabMIT

httpchop

chopcb

uuibno

SelectCR

ISPR

targetsites

andpredicto

ff-targetsites

with

Arabidopsis

geno

me[119

]University

ofBe

rgen

httppo

rtalsb

roadinstituteorggpp

pub

licanalysis

-toolssgrna-desig

nDesignhigh

lyactiv

esgR

NAs

forthe

provided

targets

[74]

BroadInstituteof

MIT

andHarvardC

ambridge

University

httpeend

bzfgenetic

sorgcasot


ndingpo


ofanyuser-specific

geno

me[120]

Peking

University

httpsgroup

sgoo

glec

omfo

rumfo

rumcris

prAc

tived

iscussio

ngrou

psGoo

gle





Abbreviations



Competing Interests


Acknowledgments


References






































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Table1Usefulresou

rcesto

olsfor

CRISPR

-Cas9research

inplants

Site

Purpose

Authority

httpwwwaddgeneo

rgAc

cessto

plasmid

resource

andtutoria

ldocum

ents[115]

Addgene

httpsw

wwprotocolsio

Accessto


Protocolsio

httpcbih


RDesignop

timalsgRN

Awith

43plantgenom

esfro

mEn

semblPlants[24]

Huazhon

gAgricultural

University

httpwwwgeno

mea

rizon

aedu

cris

prCRISP

Rsearchhtml

Predicth

ighspecifics

gRNAof

8plantgenom

es[116]

University

ofAriz

ona

httpwwwrgenom

enetcas-offind

er

Search

forp

otentia

loff-targetsites

ofsgRN

Afro

m37

plantgenom

es[117]

InstituteforB

asic

ScienceKo

rea

httpwwwe-crisp

orgE-C

RISP

indexhtml

DesignsgRN

Aforg

enom

e-lib

rarie

sprojectso

rindividu

alsequ

encesw

ith11plantgenom

es[118]

German

Cancer

Research

Center(DKF

Z)

httpcrisp

rmitedu

Find

theC

RISP

R-Ca

s9targetsites

with

inan

inpu

tsequ

ence

with

Arabidopsis

geno

me[24]

ZhangLabMIT

httpchop

chopcb

uuibno

SelectCR

ISPR

targetsites

andpredicto

ff-targetsites

with

Arabidopsis

geno

me[119

]University

ofBe

rgen

httppo

rtalsb

roadinstituteorggpp

pub

licanalysis

-toolssgrna-desig

nDesignhigh

lyactiv

esgR

NAs

forthe

provided

targets

[74]

BroadInstituteof

MIT

andHarvardC

ambridge

University

httpeend

bzfgenetic

sorgcasot


ndingpo


ofanyuser-specific

geno

me[120]

Peking

University

httpsgroup

sgoo

glec

omfo

rumfo

rumcris

prAc

tived

iscussio

ngrou

psGoo

gle





Abbreviations



Competing Interests


Acknowledgments


References






































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Abbreviations



Competing Interests


Acknowledgments


References






































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article the power of crispr-cas9-induced genome...

Documents