CRISPR/Cas核糖核蛋白介导的植物基因组编辑

doi:10.16288/j.yczz.20-017

[an error occurred while processing this directive]

Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (6): 556-564.doi: 10.16288/j.yczz.20-017

• Review • Previous Articles Next Articles

Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes

Xia Li, Wan Shi, Lizhao Geng, Jianping Xu()

Syngenta Beijing Innovation Center, Beijing 102206, China

Received:2020-01-13 Revised:2020-04-27 Online:2020-06-20 Published:2020-05-21
Contact: Xu Jianping E-mail:jianping.xu@syngenta.com

Abstract

Abstract:

The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.

Key words: CRISPR/Cas, genome editing, ribonucleoprotein (RNP), DNA-free, plant transformation

Xia Li, Wan Shi, Lizhao Geng, Jianping Xu. Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes[J]. Hereditas(Beijing), 2020, 42(6): 556-564.

Figures/Tables 2

Fig. 1

Table 1

Studies of CRISPR/Cas RNP-mediated plant genome editing"

性状基因	细胞类型	导入方法	编辑工具	编辑结果	文献来源
油菜素内酯信号途径负调控因子基因(brassinosteroid insensitive 2, BIN2)的同源基因	生菜(L. sativa)原生质体Lactuca sativa	PEG	Cas9	编辑体占再生植株的46%	[6]
茉莉酸合成途径丙二烯氧化物环化酶基因(allen oxide cyclase, AOC)	烟草(Nicotiana tabacum L.)原生质体	PEG	Cas9	深度测序编辑频率44%	[6]
光敏色素B基因(phytochrome B, PHYB),油菜素内酯信号途径负调控因子基因(brassinosteroid insensitive 1, BRI1)	拟南芥(Arabidopsis thaliana L. Heynh.) 原生质体	PEG	Cas9	PHYB：深度测序编辑频率16%;BRI1：两条sgRNA同时引导切割,在两个靶点间产生223 bp缺失	[6]]
细胞色素基因P450,DDB1结合WD40蛋白编码基因DWD1	水稻(O. sativa cv. Dongjin)原生质体	PEG	Cas9	深度测序编辑频率：19%(P450)和8.4%(DWD1)	[6]]
硝酸还原酶基因(nitrate reductase, NR)	矮牵牛(Petunia × hybrida hort. ex E. Vilm.)原生质体	PEG	Cas9	深度测序编辑频率5.3%~17.8%	[39]]
苹果火疫病易感基因DIPM-1,2,4	苹果(Malus domestica Borkh. cv. Golden delicious)原生质体	PEG	Cas9	深度测序编辑频率：6.7%(DIPM-1),0.5-3.3%(DIPM-2),2.5-6.9%(DIPM-3)	[40]]
葡萄白粉病易感基因(mildew resistance locus O-7, MLO-7)	葡萄(V. vinifera cv. Chardonnay)原生质体	PEG	Cas9	深度测序编辑频率0.1%	[40]
叶舌发育相关基因(ligulelss1, LIG1),乙酰乳酸合成酶基因(acetolactate synthase 2, ALS2),雄性不育基因MS26、MS45	玉米(Z. mays L.)幼胚	基因枪加阳离子脂质体	Cas9	编辑体分别占再生植株的4.0%(MS45)、9.7%(LIG1)和2.4%(MS26);两棵植株在ALS2位点发生同源重组介导的氨基酸精准替换	[26]
籽粒粒重相关基因(grain width 2, TaGW2),籽粒粒长相关基因TaGASR7	小麦(T. aestivum cv. Kenong 199和cv. YZ814)幼胚	基因枪	Cas9	编辑体分别占总外植体的4%~5%(cv. Kenong 199)和1%~2%(cv. YZ814)	[30]
脂肪酸去饱和酶基因(fatty acid desaturase, FAD)2-1A、FAD2-1B	大豆(Glycine max L. Merr.)原生质体	PEG	Cas12a	深度测序编辑频率：FAD2-1A 0.0%~11.7%,FAD2-1B 0.0%~9.1%	[41]
丙二烯氧化物环化酶基因AOC	野生烟草(Nicotiana attenuata Torr. ex S.Watson)原生质体	PEG	Cas12a	深度测序编辑频率0.08%~0.8%	[41]
类胡萝卜素合成途径八氢番茄红素脱氢酶基因(phytoene desaturase, PDS)和春化作用调控基因(frigida ,FRI)	卷心菜(Brassica oleracea var. capitata L. f. alba)、大白菜(B. rapa subsp. pekinensis (Lour.) Hanelt ex Mansf.)和油菜(B. napus L. cv. Topaz)原生质体	PEG	Cas9	深度测序编辑频率：卷心菜0.09%~2.25%(FRI)和0.14%~ 1.33% (PDS),大白菜1.15%~ 24.51%(FRI)和3.78%~24.51% (PDS),油菜0%	[42]
结合态淀粉合成酶基因(granule-bound starch synthase, GBSS)	马铃薯(S. tuberosum cv. Kuras)原生质体	PEG	Cas9	编辑体占再生植株的：1%~9% (化学合成sgRNA)和22%~ 25% (体外转录sgRNA);4条等位基因全部被编辑的植株占再生植株的2%~3%	[43]
红色荧光蛋白报告基因DsRed2,中脉形成和心皮发育调控基因(drooping leaf, DL),籽粒粒形相关基因(grain width 7, GW7),精卵融合调控基因(generative cell specific-1, GCS1)	水稻(O. sativa cv. Nipponbare)合子	PEG	Cas9	编辑体植株分别占PEG转化合子数的25% (DsRed2)、13.6%~14.3% (DL)、21.4% (GW7)和64% (GCS1)	[46]
八氢番茄红素脱氢酶基因OsPDS	水稻(O. sativa cv. Nipponbare)幼胚	基因枪	Cas9	编辑体占再生植株(经共转化筛选标记筛选)的33.8%	[8]
八氢番茄红素脱氢酶基因OsPDS	水稻(O. sativa cv. Nipponbare)幼胚	基因枪	Cas9和Cas12a	编辑体分别占再生植株(经共转化筛选标记筛选)的3.6%(野生型Cas9)、8.8% (HiFi Cas9)、0.0% (AsCas12a)和23.8% (LbCas12a)	[31]
橙色荧光蛋白报告基因pporRFP	烟草(N. tabacum cv. Bright Yellow-2)BY-2悬浮细胞系	阳离子脂质体和基因枪	Cas9	编辑体占随机挑选细胞团的6% (阳离子脂质体)和3% (基因枪)	[48]

Table 1

References 60

[1]	Liu PF, Wu Q . Probing 3D genome by CRISPR/Cas9. Hereditas(Beijing), 2020,42(1):18-31.
	刘沛峰, 吴强 . CRISPR/Cas9基因编辑在三维基因组研究中的应用. 遗传, 2020,42(1):18-31.
[2]	Voytas DF, Gao CX . Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol, 2014,12(6):e1001877.
[3]	Jones HD . Regulatory uncertainty over genome editing. Nat Plants, 2015,1:14011.
[4]	Chen K, Wang Y, Zhang R, Zhang H, Gao C . CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol, 2019,70:667-697.
[5]	Metje-Sprink J, Menz J, Modrzejewski D, Sprink T . DNA-free genome editing: past, present and future. Front Plant Sci, 2018,9:1957.
[6]	Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS . DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol, 2015,33(11):1162-1164.
[7]	Modzelewski AJ, Chen S, Willis BJ, Lloyd KCK, Wood JA, He L . Efficient mouse genome engineering by CRISPR- EZ technology. Nat Protoc, 2018,13(6):1253-1274.
[8]	Banakar R, Eggenberger AL, Lee K, Wright DA, Murugan K, Zarecor S, Lawrence-Dill CJ, Sashital DG, Wang K . High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice. Sci Rep, 2019,9(1):19902.
[9]	Koonin EV, Makarova KS . CRISPR-Cas: an adaptive immunity system in prokaryotes. F1000 Biol Rep, 2009,1:95.
[10]	Shan QW, Gao CX . Research progress of genome editing and derivative technologies in plants. Hereditas(Beijing), 2015,37(10):953-973.
	单奇伟, 高彩霞 . 植物基因组编辑及衍生技术最新研究进展. 遗传, 2015,37(10):953-973.
[11]	Gasiunas G, Barrangou R, Horvath P, Siksnys V . Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA, 2012,109(39):E2579-2586.
[12]	Adli M . The CRISPR tool kit for genome editing and beyond. Nat Commun, 2018,9(1):1911.
[13]	Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F . Multiplex genome engineering using CRISPR/Cas systems. Science, 2013,339(6121):819-823.
[14]	Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015,163(3):759-771.
[15]	Tang LC, Gu F . Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM. Hereditas (Beijing), 2020,42(3):236-249.
	唐连超, 谷峰 . CRISPR-Cas基因编辑系统升级:聚焦Cas蛋白和PAM. 遗传, 2020,42(3):236-249.
[16]	Makarova KS, Zhang F, Koonin EV. SnapShot: Class 2 CRISPR-Cas Systems. Cell, 2017,168(1-2): 328-328.e321.
[17]	Yao RL, Liu D, Jia X, Zheng Y, Liu W, Xiao Y . CRISPR- Cas9/Cas12a biotechnology and application in bacteria. Synth Syst Biotechnol, 2018,3(3):135-149.
[18]	Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821.
[19]	Li J, Zhang Y, Chen KL, Shan QW, Wang YP, Liang Z, Gao CX . CRISPR/Cas: a novel way of RNA-guided genome editing. Hereditas(Beijing), 2013,35(11):1265-1273.
	李君, 张毅, 陈坤玲, 单奇伟, 王延鹏, 梁振, 高彩霞 . CRISPR/Cas系统:RNA靶向的基因组定向编辑新技术. 遗传, 2013,35(11):1265-1273.
[20]	Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA . DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 2014,507(7490):62-67.
[21]	Li SY, Li JY, He YB, Xu ML, Zhang JH, Du WM, Zhao YD, Xia LQ . Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat Biotechnol, 2019,37(4):445-450.
[22]	Li B, Rui HP, Li YJ, Wang QQ, Alariqi M, Qin L, Sun L, Ding X, Wang FQ, Zou JW, Wang YQ, Yuan DJ, Zhang XL, Jin SX . Robust CRISPR/Cpf1 (Cas12a)-mediated genome editing in allotetraploid cotton (Gossypium hirsutum). Plant Biotechnol J, 2019,17(10):1862-1864.
[23]	Malzahn AA, Tang X, Lee K, Ren QR, Sretenovic S, Zhang YX, Chen HQ, Kang M, Bao Y, Zheng XL, Deng KJ, Zhang T, Salcedo V, Wang K, Zhang Y, Qi YP . Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol, 2019,17(1):9.
[24]	Tu M, Lin L, Cheng Y, He X, Sun H, Xie H, Fu J, Liu C, Li J, Chen D, Xi H, Xue D, Liu Q, Zhao J, Gao C, Song Z, Qu J, Gu F . A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res, 2017,45(19):11295-11304.
[25]	Tu ZC, Yang WL, Yan S, Yin A, Gao JQ, Liu XD, Zheng YH, Zheng JZ, Li ZJ, Yang S, Li SH, Guo XY, Li XJ . Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci Rep, 2017,7:42081.
[26]	Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A . Genome editing in maize directed by CRISPR- Cas9 ribonucleoprotein complexes. Nat Commun, 2016,7:13274.
[27]	Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C . Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development, 2016,143(11):2025-2037.
[28]	Gorbunova V, Levy AA . Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res, 1997,25(22):4650-4657.
[29]	Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, Christian M, Li XH, Pierick CJ, Dobbs D, Peterson T, Joung JK, Voytas DF . High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci USA, 2010,107(26):12028-12033.
[30]	Liang Z, Chen KL, Li TD, Zhang Y, Wang YP, Zhao Q, Liu JX, Zhang HW, Liu CM, Ran YD, Gao CX . Efficient DNA-free genome editing of bread wheat using CRISPR/ Cas9 ribonucleoprotein complexes. Nat Commun, 2017,8:14261.
[31]	Banakar R, Schubert M, Collingwood M, Vakulskas C, Eggenberger AL, Wang K . Comparison of CRISPR-Cas9/ Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene. Rice (NY), 2020,13(1):4.
[32]	Cho SW, Lee J, Carroll D, Kim JS, Lee J . Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics, 2013,195(3):1177-1180.
[33]	Kim S, Kim D, Cho SW, Kim J, Kim JS . Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res, 2014,24(6):1012-1019.
[34]	McCarty NS, Graham AE, Studená L, Ledesma-Amaro R . Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat Commun, 2020,11(1):1281.
[35]	Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR . Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol, 2015,33(1):73-80.
[36]	Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD . Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol, 2015,208:44-53.
[37]	Lin S, Staahl BT, Alla RK, Doudna JA . Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife , 2014,3:e04766.
[38]	Mout R, Ray M, Yesilbag Tonga G, Lee YW, Tay T, Sasaki K, Rotello VM . Direct cytosolic delivery of CRISPR/Cas9- ribonucleoprotein for efficient gene editing. ACS Nano, 2017,11(3):2452-2458.
[39]	Subburaj S, Chung SJ, Lee C, Ryu SM, Kim DH, Kim JS, Bae S, Lee GJ . Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep, 2016,35(7):1535-1544.
[40]	Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala Kanchiswamy C . DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci, 2016,7:1904.
[41]	Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG . CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun, 2017,8:14406.
[42]	Murovec J, Guček K, Bohanec B, Avbelj M, Jerala R . DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes. Front Plant Sci, 2018,9:1594.
[43]	Andersson M, Turesson H, Olsson N, Falt AS, Ohlsson P, Gonzalez MN, Samuelsson M, Hofvander P . Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant, 2018,164(4):378-384.
[44]	Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J . Progress in plant protoplast research. Planta, 2013,238(6):991-1003.
[45]	Zhang Y, Liang Z, Zong Y, Wang YP, Liu JX, Chen KL, Qiu JL, Gao CX . Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/ Cas9 DNA or RNA. Nat Commun, 2016,7:12617.
[46]	Toda E, Koiso N, Takebayashi A, Ichikawa M, Kiba T, Osakabe K, Osakabe Y, Sakakibara H, Kato N, Okamoto T . An efficient DNA- and selectable-marker-free genome- editing system using zygotes in rice. Nat Plants, 2019,5(4):363-368.
[47]	Liang Z, Chen KL, Zhang Y, Liu JX, Yin KQ, Qiu JL, Gao CX . Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc, 2018,13(3):413-430.
[48]	Liu W, Rudis MR, Cheplick MH, Millwood RJ, Yang JP, Ondzighi-Assoume CA, Montgomery GA, Burris KP, Mazarei M, Chesnut JD, Stewart CN ,Jr. Lipofection- mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep, 2020,39(2):245-257.
[49]	Liu J, Gaj T, Patterson JT, Sirk SJ, Barbas CF . Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS One, 2014,9(1):e85755.
[50]	Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H . Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res, 2014,24(6):1020-1027.
[51]	Zorko M, Langel U . Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev, 2005,57(4):529-545.
[52]	Chugh A, Amundsen E, Eudes F . Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep, 2009,28(5):801-810.
[53]	Cunningham FJ, Goh NS, Demirer GS, Matos JL, Landry MP . Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol, 2018,36(9):882-897.
[54]	Demirer GS, Zhang H, Matos JL, Goh NS, Cunningham FJ, Sung Y, Chang R, Aditham AJ, Chio L, Cho MJ, Staskawicz B, Landry MP . High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol, 2019,14(5):456-464.
[55]	Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR . Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun, 2017,8:15790.
[56]	Yeh WH, Chiang H, Rees HA, Edge ASB, Liu DR . In vivo base editing of post-mitotic sensory cells. Nat Commun, 2018,9(1):2184.
[57]	Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH . Chemically modified guide RNAs enhance CRISPR- Cas genome editing in human primary cells. Nat Biotechnol, 2015,33(9):985-989.
[58]	Liu P, Luk K, Shin M, Idrizi F, Kwok S, Roscoe B, Mintzer E, Suresh S, Morrison K, Frazão JB, Bolukbasi MF, Ponnienselvan K, Luban J, Zhu LJ, Lawson ND, Wolfe SA . Enhanced Cas12a editing in mammalian cells and zebrafish. Nucleic Acids Res, 2019,47(8):4169-4180.
[59]	Zhang L, Vakulskas CA, Bode NM, Collingwood MA, Beltz KR, Behlke MA . Novel mutations that enhance the DNA cleavage activity of Acidaminococcus sp. Cpf1. WO 2020/033774 A1,World Intellectual Property Organization, 2020.
[60]	Serif M, Dubois G, Finoux AL, Teste MA, Jallet D, Daboussi F . One-step generation of multiple gene knock- outs in the diatom Phaeodactylum tricornutum by DNA- free genome editing. Nat Commun, 2018,9(1):3924.

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 2

References 60

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Xiaoping Lian, Guangfu Huang, Yujiao Zhang, Jing Zhang, Fengyi Hu, Shilai Zhang. The discovery and utilization of favorable genes in Oryza longistaminata [J]. Hereditas(Beijing), 2023, 45(9): 765-780.
[2]	Bingzheng Wang, Chao Zhang, Jiali Zhang, Jin Sun. Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA [J]. Hereditas(Beijing), 2023, 45(7): 593-601.
[3]	Danni Liu, Haiping Wu, Guohua Zhou. Research progress of visual detection in rapid on-site detection of pathogen nucleic acid [J]. Hereditas(Beijing), 2023, 45(4): 306-323.
[4]	Zhongsheng Wu, Yu Gao, Yongtao Du, Song Dang, Kangmin He. The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing [J]. Hereditas(Beijing), 2023, 45(2): 165-175.
[5]	Meizhen Liu, Liren Wang, Yongmei Li, Xueyun Ma, Honghui Han, Dali Li. Generation of genetically modified rat models via the CRISPR/Cas9 technology [J]. Hereditas(Beijing), 2023, 45(1): 78-87.
[6]	Xiaojun Zhang, Kun Xu, Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei. A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair [J]. Hereditas(Beijing), 2022, 44(8): 708-719.
[7]	Chong Zhang, Zixuan Wei, Min Wang, Yaosheng Chen, Zuyong He. Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis [J]. Hereditas(Beijing), 2022, 44(7): 581-590.
[8]	Yao Liu, Xianhui Zhou, Shuhong Huang, Xiaolong Wang. Prime editing: a search and replace tool with versatile base changes [J]. Hereditas(Beijing), 2022, 44(11): 993-1008.
[9]	Yuting Han, Bowen Xu, Yutong Li, Xinyi Lu, Xizhi Dong, Yuhao Qiu, Qinyun Che, Ruibao Zhu, Li Zheng, Xiaochen Li, Xu Si, Jianquan Ni. The cutting edge of gene regulation approaches in model organism Drosophila [J]. Hereditas(Beijing), 2022, 44(1): 3-14.
[10]	Haitao Wang, Tingting Li, Xun Huang, Runlin Ma, Qiuyue Liu. Application of genetic modification technologies in molecular design breeding of sheep [J]. Hereditas(Beijing), 2021, 43(6): 580-600.
[11]	Guangwu Yang, Yuan Tian. The F-box gene Ppa promotes lipid storage in Drosophila [J]. Hereditas(Beijing), 2021, 43(6): 615-622.
[12]	Dingwei Peng, Ruiqiang Li, Wu Zeng, Min Wang, Xuan Shi, Jianhua Zeng, Xiaohong Liu, Yaoshen Chen, Zuyong He. Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs [J]. Hereditas(Beijing), 2021, 43(3): 261-270.
[13]	Na Wang, Zhilian Jia, Qiang Wu. RFX5 regulates gene expression of the Pcdhα cluster [J]. Hereditas(Beijing), 2020, 42(8): 760-774.
[14]	Guoling Li, Shanxin Yang, Zhenfang Wu, Xianwei Zhang. Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals [J]. Hereditas(Beijing), 2020, 42(7): 641-656.
[15]	Yingnan Chen, Jing Lu. Application of CRISPR/Cas9 mediated gene editing in trees [J]. Hereditas(Beijing), 2020, 42(7): 657-668.