Application Progress of CRISPR/Cas9 Technology in Crop Genetic Breeding

doi:10.15933/j.cnki.1004-3268.2021.07.001

Abstract

Abstract:

CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISRP‑associated nuclease 9）is a new type of genome⁃directed editing technology after ZFNs（zinc finger nucleases）and TALENs（transcription activator like effector nucleases）.Compared with the previous two generations of technology，it has the characteristics of simplicity and high efficiency.CRISPR/Cas9 is not only a basic research tool，but also has become one of currently useful molecular breeding tools，and important progress has been made in crop genetic improvement. The structure，classification，action mechanism of the CRISPR/Cas9 system and its application progress in crop quality improvement，yield enhancement，resistance breeding and male sterile material selection were reviewed，its existing problems were discussed，and its application prospects were prospected.

Key words: ">CRISPR/Cas9；Genome editing；gRNA；Crop；Genetic breeding

摘要： CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISRP‑associated nuclease 9)是继ZFNs(Zinc finger nucleases)和TALENs(Transcription activator like effector nucleases)之后出现的新型基因组定点编辑技术，相较于前两代技术，具有简便、高效等特点。CRISPR/Cas9不仅是一种基础性研究工具，而且已经成为目前有用的分子育种工具之一，在作物遗传改良方面取得重要应用进展。综述了CRISPR/Cas9系统的结构、分类、作用机制及在作物品质改良、产量提升、抗性育种和雄性不育材料选育中的应用进展，并探讨了其存在的问题，展望了其应用前景。

关键词: CRISPR/Cas9, 基因组编辑, gRNA, 作物, 遗传育种

CLC Number:

S158

JIAO Yaolei, WANG Chunsheng, QU Shuo, SUN Shanshan, ZHU Tingting, ZHAO He, WANG Piwu. Application Progress of CRISPR/Cas9 Technology in Crop Genetic Breeding[J]. Journal of Henan Agricultural Sciences, 2021, 50(7): 1-7.

焦耀磊, 王春生, 曲硕, 孙珊珊, 朱婷婷, 赵贺, 王丕武. CRISPR/Cas9 技术在作物遗传育种中的应用进展[J]. 河南农业科学, 2021, 50(7): 1-7.

References

参考文献：
［1］ SUWEIS S，CARR J A，MARITAN A，et al. Resilience and reactivity of global food security［J］. Proceedings of the National Academy of Sciences，2015，112（22）：6092⁃6097.
［2］ CHEESEMAN J. Food security in the face of salinity，drought，climate change，and population growth［M］//KHAN M A，OZTURK M，GUL B，et al. Halophytes for food security in dry lands. Amsterdam：Elsevier⁃Academic Press，2016:111⁃123.
［3］ WU Q H，ZHANG S X，FENG G，et al. Determining the optimum range of soil olsen P for high P use efficiency，crop yield，and soil fertility in three typical cropland soils［J］. Pedosphere，2020，30（6）：832⁃843.
［4］ LIU H，DING Y，ZHOU Y，et al. CRISPR⁃P 2. 0：Animproved CRISPR⁃Cas9 tool for genome editing in plants［J］. Molecular Plant，2017，10（3）：530⁃533.
［5］ SOREK R，LAWRENCE C M，WIEDENHEFT B.CRISPR⁃mediated adaptive immune systems in bacteria and archaea［J］. Annual Review of Biochemistry，2013，82（1）：237⁃266.
［6］ CARROLL D. Genome engineering with zinc⁃finger nucleases［J］. Genetics，2011，188（4）：773⁃782.
［7］ MILLER J C，HOLMES M C，WANG J，et al. An improved zinc⁃fifinger nuclease architecture for highly specific genome editing［J］. Nat Biotechnol，2007，25（7）：778⁃785.
［8］ SANDER J D，DAHLBORG E J，GOODWIN M J，et al.Selection⁃free zinc⁃finger⁃nuclease engineering by context⁃dependent assembly（CoDA）［J］. Nature Methods，2011，8（1）：67⁃69.
［9］ BOGDANOVE A J，VOYTAS D F. TAL Effectors：Customizable proteins for DNA targeting［J］. Science，2011，333：1843⁃1846.
［10］ CERMAK T，DOYLE E L，CHRISTIAN M，et al.Efficient design and assembly of custom TALEN and other TAL effector⁃based constructs for DNA targeting［J］. Nucleic Acids Research，2011，39（12）：82⁃83.
［11］ CHEN K，GAO C. TALENs：Customizable molecular DNA scissors for genome engineering of plants［J］.Journal of Genetics and Genomics，2013，40（6）：271⁃279.
［12］ STREUBEL J，BLÜCHER C，LANDGRAF A，et al.TAL effector RVD specificities and efficiencies［J］.Nature Biotechnology，2012，30（7）：593⁃595.
［13］ LI T，LIU B，SPALDING M H. High⁃efficiency TALEN⁃based gene editing produces disease⁃resistant rice［J］. Nat Biotechnol，2012，30（5）：390⁃392.
［14］ SHUKLA V K，DOYON Y，MILLER J C，et al. Precise genome modification in the crop species Zea mays using zinc⁃finger nucleases［J］. Nature，2009，459：437⁃474.
［15］ TOWNSEND J A，WRIGHT D A，WINFRERJ Y，et al.High frequency modification of plant genes using engineered zinc finger nucleases［J］. Nature，2009，459：442⁃454.
［16］ ZHANG F，MAEDER M L，UNGER⁃WALLACE E，et al. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases［J］.Proceedings of the National Academy of Sciences，2010，107（26）：12028⁃12033.
［17］ ISHINO Y，SHINAGAWA H，MAKINO K，et al.Nucleotide sequence of the iap gene，responsible for alkaline phosphatase isozyme conversion in Escherichia coli，and identification of the gene product［J］. Journal of Bacteriology，1987，169（12）：5429⁃5433.
［18］ MOJICA F J M， DÍEZ⁃VILLASEÑOR C，GARCÍA⁃MARTÍNEZ J，et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements［J］. Journal of Molecular Evolution，2005，60（2）：174⁃182.
［19］ GUY C P，MAJERNÍK A I，CHONG J P. A novel nuclease⁃ATPase（Nar71）from archaea is part of a proposed thermophilic DNA repair system［J］. Nucleic Acids Research，2004，32（21）：6176⁃6186.
［20］ MAKAROVA K S，ARAVIND L，GRISHIN N V. A DNA repair system specifific for thermophilic archaea and bacteria predicted by genomic context analysis［J］.Nucleic Acids Research，2002，30（2）：482⁃496.
［21］ JANSEN R，VAN E J D A，GAASTERA W，et al.Identification of a novel family of sequence repeats among prokaryotes［J］. Omics，2002，6（1）：23⁃33.
［22］ BOLOTIN A. Clustered regularly interspaced short palindrome repeats （CRISPRs） have spacers of extrachromosomal origin［J］. Microbiology，2005，151（8）：2551⁃2561.
［23］ HAFT D H，SELENGUT J，MNOGODIN E F. A guild of 45 CRISPR⁃associated（Cas）protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes［J］.PLoS Computational Biology，2005，1（6）：60⁃65.
［24］ JANSEN R，EMBDEN J D V A，GAASTRA W，et al.Identification of genes that are associated with DNA repeats in prokaryotes［J］. Molecular Microbiology，2002，43（6）：1565⁃1575.
［25］ MAKAROVA K S，GRISHIN N，SHABALINA S A. A putative RNA⁃interference⁃based immune system in prokaryotes：Computational analysis of the predicted enzymatic machinery， functional analogies with eukaryotic RNAi，and hypothetical mechanisms of action［J］. Biology Direct，2006，1（1）：7⁃8.
［26］ BARRANGOU R，FREMAUX C，DEVEAU H，et al.CRISPR provides acquired resistance against viruses in prokaryotes［J］. Science，2007，315：1709⁃1712.
［27］ BARRANGOU R，HORVATH P. CRISPR：New horizons in phage resistance and strain identification［J］. Annual Review of Food Science and Technology，2012，3（1）：143⁃162.
［28］ CONG L，RAN F A，COX D，et al. Multiplex genome engineering using CRISPR/Cas systems［J］. Science，2013，339：819⁃823.

［29］ ANDRIY B，DANIEL G，JOHN L. Emerging genome engineering tools in crop research and breeding［J］.Methods in Molecular Biology，2020，2072：165⁃181.

［30］ HALE C R，MAJUMDAR S，ELMORE J，et al.Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs［J］. Molecular Cell，2012，45（3）：292⁃302.

［31］ MAKAROVA K S，HAFT D H，BARRANGOU R，et al.Evolution and classification of the CRISPR⁃Cas systems［J］. Nature Reviews Microbiology，2011，9（6）：467⁃477.
［32］ GONG B，SHIN M，SUN J L，et al. Molecular insights into DNA interference by CRISPR⁃associatednuclease⁃helicase Cas3［J］. Proceedings of the National Academy of Sciences of the United States of America，2014，111（46）：16359⁃16364.
［33］ SINKUNAS T，GASIUNAS G，FREMAUX C，et al.Cas3 is a single⁃stranded DNA nuclease and ATP⁃dependent helicase in the CRISPR/Cas immune system［J］. The EMBO Journal，2011，30（7）：1335⁃1342.
［34］ CHYLINSKI K，RHUN A L，CHARPENTIER E. The tracrRNA and Cas9 families of type Ⅱ CRISPR⁃Cas immunity systems［J］. RNA Biology，2013，10（5）：726⁃737.
［35］ KRZYSZTOF C，MAKAROVA K S，EMMANUELLE C，et al. Classification and evolution of type Ⅱ CRISPR⁃Cas systems［J］. Nucleic Acids Research，2014，42（10）：6091⁃6105.
［36］ HUO Y，NAM K H，DING F，et al. Structures of CRISPR Cas3 offer mechanistic insights into cascade⁃activated DNA unwinding and degradation［J］.Nature Structural & Molecular Biology，2014，21（9）：771⁃777.
［37］ BRINER A E，DONOHOUE P D，GOMAA A A，et al.Guide RNA functional modules direct Cas9 activity and orthogonality［J］. Molecular Cell，2014，56（2）：333⁃339.
［38］ TAMULAITIS G，KAZLAUSKIENE M，MANAKOVAE，et al. Programmable RNA shredding by the type Ⅲ：A CRISPR⁃Cas system of Streptococcus thermophilus［J］. Molecular Cell，2014，56（4）：506⁃517.
［39］ HELER R，MARRAFFINI L A，BIKARD D. Adapting to new threats： The generation of memory by CRISPR⁃Cas immune systems ［J］. Molecular Microbiology，2014，93（1）：1⁃9.
［40］ MARRAFFINI L A. CRISPR⁃Cas immunity in prokaryotes［J］. Nature，2015，526：55⁃61.

［41］ MOJICA F J M，RODRIGUEZ⁃VALERA F. The discovery of CRISPR in archaea and bacteria［J］. The FEBS Journal，2016，283（17）：3162⁃3169.

［42］ AMITAI G，SOREK R. CRISPR⁃Cas adaptation：Insights into the mechanism of action［J］. Nature Reviews Microbiology，2016，14（2）：67⁃76.

［43］ COCOZAKI A，RAMIA N，SHAO Y，et al. Structure of the Cmr2 subunit of the CRISPR⁃Cas RNA silencing complex［J］. Structure，2012，20（3）：545⁃553.
［44］ BROUNS S J J，JORE M M，LUNDGREN M. Small CRISPR RNAs guide antiviral defense in prokaryotes［J］. Science，2008，321：960⁃962.
［45］ JIANG F，DOUDNA J A. The structural biology of CRISPR⁃Cas systems［J］. Current Opinion in Structural Biology，2015，30（1）：100⁃111.
［46］ WIEDENHEFT B，STERNBERG S H，DOUDNA J A.RNA⁃guided genetic silencing systems in bacteria and archaea［J］. Nature，2012，482：331⁃338.
［47］ MARTIN M，FITZGERALD M A. Proteins in rice grains influence cooking properties！［J］. Journal of Cereal Science，2002，36（3）：285⁃294.
［48］ BALINDONG J L，WARD R M，LEI L，et al. Rice grain protein composition influences instrumental measures of rice cooking and eating quality［J］.Journal of Cereal Science，2018，79（2）：35⁃42.
［49］ DINKELOO K，BOYD S，PILOT G. Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants［J］. Seminars in Cell and Developmental Biology，2018，74（2）：105⁃113.
［50］ WANG S Y，YANG Y H，GUO M，et al. Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system［J］. The Crop Journal，2020，8（3）：457⁃464.
［51］ LI Y，LIU D，ZONG Y，et al. New D hordein alleles were created in barley using CRISPR/Cas9 genome
editing［J］. Cereal Research Communications，2020，48（2）：131⁃138.
［52］ WANG H，WU Y，ZHANG Y，et al. CRISPR/Cas9⁃based mutagenesis of starch biosynthetic genes in sweet
potato （Ipomoea batatas） for the improvement of starch quality［J］. International Journal of Molecular Sciences，2019，20（19）：4702⁃4705.
［53］ HUANG H，CUI T，ZHANG L，et al. Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9⁃mediated gene editing in Brassica napus［J］. Theoretical and Applied Genetics，2020，133（8）：2401⁃2411.
［54］沈兰，李健，付亚萍，等. 利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状［J］. 中国水稻科学，2017，31（3）：223⁃231.
SHEN L，LI J，FU Y P，et al. Orientation improvement of grain length and grain number in rice by using CRISPR/Cas9 system［J］. Chinese Journal of Rice Science，2017，31（3）：223⁃231.
［55］ ZHOU J P，XIN X H，HE Y，et al. Multiplex QTL editing of grain⁃related genes improves yield in elite rice varieties［J］. Plant Cell Reports，2019，38（4）：475⁃485.

［56］张笑寒. CRISPR/Cas9定点编辑OsGA20ox2基因降低水稻株高研究［D］. 贵阳：贵州大学，2016.

ZHANG X H. Mutation of OsGA20ox2 gene based on CRISPR/Cas9 editing system in rice［D］. Guiyang： Guizhou University，2016.

［57］ ZAMAN Q U，CHU W，HAO M，et al. CRISPR/Cas9⁃mediated multiplex genome editing of JAGGED gene in Brassica napus L［J］. Biomolecules，2019，9（11）：725⁃729.
［58］ TAKAGI H，TAMIRU M，ABE A，et al. MutMap accelerates breeding of a salt⁃tolerant rice cultivar［J］.Nature Biotechnology，2015，33（5）：445⁃459.
［59］ ZHANG A，LIU Y，WANG F，et al. Enhanced rice salinity tolerance via CRISPR/Cas9⁃targeted mutagenesis
of the OsRR22 gene［J］. Molecular Breeding，2019，39（3）：1⁃10.
［60］ LIU L，ZHANG J L，XU J Y，et al. CRISPR/Cas9 targeted mutagenesis of SlLBD40，a lateral organ boundaries domain transcription factor， enhances drought tolerance in tomato［J］. Plant Science，2020，301（2）：1⁃13.
［61］郝巍. 利用CRISPR/Cas9技术对水稻白叶枯病感病相关基因HW3进行定点修饰及功能分析［D］. 北京：中国农业科学院，2016.
HAO W. Identification of a susceptibility⁃related gene HW3 for bacterial blight of rice by using CRISPR/Cas9 system［D］. Beijing：Chinese Academy of Agricultural Sciences，2016.
［62］ WANG F J，WANG C L，LIU P Q，et al. Enhanced rice blast resistance by CRISPR/Cas9⁃targeted mutagenesis
of the ERF transcription factor gene OsERF922［J］.PLoS One，2016，11（4）：1⁃13.
［63］ LI J，WANG Z，HE G G，et al. CRISPR/Cas9⁃mediated disruption of TaNP1 genes results in complete male
sterility in bread wheat［J］. Journal of Genetics and Genomics，2020，47（5）：263⁃272.
［64］刘玉琛，丘式浚，金曼，等. CRISPR/Cas9技术在创制番茄雄性不育株系中的应用研究［J］. 农业生物技术学报，2019，27（6）：951⁃960.
LIU Y C，QIU S J，JIN M，et al. Study on the application of CRISPR/Cas9 techonlogy in development of tomato
（Solanum lycopersicum）male sterile line［J］. Journal of Agricultural Biotechnology，2019，27（6）：951⁃960.
［65］ SHEN L，DONG G J，ZHANG Y，et al. Rapid creation of new photoperiod⁃/thermo⁃sensitive genic male⁃sterile
rice materials by CRISPR/Cas9 system［J］. Rice Science，2019，26（2）：129⁃132.
［66］ CHEN R G，XU Q L，LIU Y. Generation of transgene⁃free maize male sterile lines using the
CRISPR/Cas9 system［J］. Frontiers in Plant Science，2018，9（7）：1180⁃1196.

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[14]	. [J]. Journal of Henan Agricultural Sciences, 2018, 47(2): 22-28.
[15]	. [J]. Journal of Henan Agricultural Sciences, 2009, 38(11): 64-68.