一个CRISPR/Cas9-VQR基因编辑系统的构建

doi:10.3724/SP.J.1006.2019.82052

摘要/Abstract

摘要：

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术, 但该编辑系统的使用范围受PAM (proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9 (SpCas9)编码氨基酸 (1135位的天冬氨酸D突变成缬氨酸V, 1335位的精氨酸R突变为谷胱氨酸Q, 1337位的苏氨酸T突变为精氨酸R, 命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的 3′ UTR 序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点, 并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%~70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%~70.5%, 平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围, 特别是NGA PAM位点较高的作物。

关键词: CRISPR/Cas9-VQR, 定点突变, PAM, 基因突变

Abstract:

Clustered Regularly Interspaced Short Palindromic Repeat and Cas9 (CRISPR/Cas9), a new generation of genome- editing technology, is widely applied among bacteria, yeast, animals and plants, however, the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif (PAM), which limits its application. In order to broaden the applications of CRIPSR/Cas9 system, we modified the Streptococcus pyogenes Cas9 (SpCas9) sequence by the PCR site-direct mutagenesis, which encodes V (1135), Q (1335), and R (1337), to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif. We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR, optimizing SpCas9-codon, adding conserved nuclear localization signal sequence, and increasing the conserved 3' UTR sequence of monocots, and using OsU6 transcripts of sRNA. CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo. We assembled the Cas9-VQR protein with the sRNAs in vitro. The Cas9-VQR could cleave the targeted fragments with about 5%-70% of mutation efficiency. In the transformation of rice, we detected about 27.50%-70.50% of mutation ratio, with an average of 46.23%. This system broadens the CRISPR/Cas9 applications in crops, especially in these with higher PAM locus of NGA.

Key words: CRISPR/Cas9-VQR, site-direct mutagenesis, PAM, mutation rate

陈凯,孙国梁,宋高原,李爱丽,谢传晓,毛龙,耿帅锋. 一个CRISPR/Cas9-VQR基因编辑系统的构建[J]. 作物学报, 2019, 45(6): 848-855.

Kai CHEN,Guo-Liang SUN,Gao-Yuan SONG,Ai-Li LI,Chuan-Xiao XIE,Long MAO,Shuai-Feng GENG. Establishment of a CRISPR/Cas9-VQR gene editing system[J]. Acta Agronomica Sinica, 2019, 45(6): 848-855.

图/表 10

表1

表2

表3

图1

图2

图3

图4

图5

图6

表5

参考文献 41

[1]	Song G Y, Jia M L, Chen K, Kong X C, Khattak B, Xie C X, Li A L, Mao L . CRISPR/Cas9: A powerful tool for crop genome editing. Crop J, 2016,4:75-82. doi: 10.1016/j.cj.2015.12.002
[2]	Shukla V K, Doyon Y, Miller J C , DeKelver R C, Moehle E A, Worden S E, Mitchell J C, Arnold N L, Gopalan S, Meng X, Choi V M, Rock J M, Wu Y Y, Katibah G E, Zhifang G, McCaskill D, Simpson M A, Blakeslee B, Greenwalt S A, Butler H J, Hinkley S J, Zhang L, Rebar E J, Gregory P D, Urnov F D . Precise genome modification in the crop speciesZea mays using zinc-finger nucleases. Nature, 2009,459:437-441. doi: 10.1038/nature07992 pmid: 19404259
[3]	Schiml S, Fauser F, Puchta H . The CRISPR/Cas9 system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J, 2014,80:1139-1150. doi: 10.1111/tpj.12704 pmid: 25327456
[4]	Shukla V K, Doyon Y, Miller J C , DeKelver R C, Moehle E A, Worden S E, Mitchell J C, Arnold N L, Gopalan S, Meng X, Choi V M, Rock J M, Wu Y Y, Katibah G E, Zhifang G, McCaskill D, Simpson M A, Blakeslee B, Greenwalt S A, Butler H J, Hinkley S J, Zhang L, Rebar E J, Gregory P D, Urnov F D. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 2009,459:437-411.
[5]	Fauser F, Schiml S, Puchta H . Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J, 2014,2:348-359. doi: 10.1111/tpj.12554 pmid: 24836556
[6]	Li J F, Norville J E, Aach J , McCormack M, Zhang D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol, 2013,31:688-691.
[7]	Fauser F, Schiml S, Puchta H . Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J, 2014,79:348-359. doi: 10.1111/tpj.12554 pmid: 24836556
[8]	Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J K . Efficient genome editing in plants using a CSRIPR/Cas system. Cell Res, 2013,23:1229-1232.
[9]	Hyun Y B, Kim J, Cho S W, Choi Y, Kim J S, Coupland G . Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted REGN of the CRISPR/Cas9 system to generate heritable null alleles. Planta, 2015,241:271-284.
[10]	Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D L, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu J K . Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA, 2014,111:4632-4637.
[11]	Ali Z , Abul-faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes N , Voytas D F, Dinesh-Kumar S, Mahfouz M M. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant, 2015,8:1288-1291. doi: 10.1016/j.molp.2015.02.011 pmid: 25749112
[12]	Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, Wu Y, Zhao P, Xia Q . CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol, 2015,87:99-110.
[13]	Nekrasov V, Staskawicz B, Weigel D, Jones J D, Kamoun S . Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013,31:691-693.
[14]	Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C . Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol, 2013,31:686-688. doi: 10.1038/nbt.2650 pmid: 23929338
[15]	Miao J, Guo D S, Zhang J Z, Huang Q P, Qin G J, Zhang X, Wan J M, Gu H Y, Qu L J . Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res, 2013,23:1233-1236. doi: 10.1038/cr.2013.123 pmid: 23999856
[16]	Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu J K . The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J, 2014,12:797-807. doi: 10.1111/pbi.12200 pmid: 24854982
[17]	Xie K B, Yang Y N . RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant, 2013,6:1975-1983. doi: 10.1093/mp/sst119 pmid: 23956122
[18]	Xu R, Li H, Qin R, Wang L, Li L, Wei P, Yang J . Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice, 2014,1:5-7.
[19]	Xie K B, Minkenberg B, Yang Y N . Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA, 2015,112:3570-3575. doi: 10.1073/pnas.1420294112 pmid: 25733849
[20]	Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y G . A robust CRISPR/Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015,8:1274-1284. doi: 10.1016/j.molp.2015.04.007 pmid: 25917172
[21]	Zhou H, Liu B, Weeks D P, Spalding M H, Yang B . Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res, 2014,42:10903-10914. doi: 10.1093/nar/gku806 pmid: 4176183
[22]	Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y . Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep, 2015,5:10342. doi: 10.1038/srep10342 pmid: 26022141
[23]	Pan C T, Ye L, Qin L, Liu X, He Y J, Wang J, Chen L F, Lu G . CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep, 2016,6:24765. doi: 10.1038/srep24765 pmid: 27097775
[24]	Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks D P . Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res, 2013,41:e188.
[25]	Liang Z, Zhang K, Chen K, Gao C . Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genomics, 2014,41:63-68. doi: 10.1016/j.jgg.2013.12.001 pmid: 24576457
[26]	Li C, Unver T, Zhang B H . A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton (Gossypium hirsutum L.). Sci Rep, 2017,7:43902. doi: 10.1038/srep43902 pmid: 5335549
[27]	Shan Q W, Wang Y P, Li J, Gao C X . Genome editing in rice and wheat using the CRISPR/Cas9 system. Nat Protocol, 2014,9:2395-2410. doi: 10.1038/nprot.2014.157 pmid: 25232936
[28]	Li B, Cui G, Shen G, Zhan Z, Huang L, Chen J, Qi X . Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza. Sci Rep, 2017,7:43320.
[29]	Xu R, Li H, Qin R, Wang L, Li L, Wei P, Yang J . Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice, 2014,7:1-5.
[30]	Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu J K . Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant, 2013,6:2008-2011 doi: 10.1093/mp/sst121 pmid: 23963532
[31]	Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J . A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 2014,14:327-328. doi: 10.1186/s12870-014-0327-y pmid: 4262988
[32]	Li J F, Norville J E, Aach J , McCormack M, Zhang D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol, 2013,8:688.
[33]	Liu W, Zhu X, Lei M, Xia Q, Botella J R, Zhu J K, Mao Y . A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana. Sci Bull, 2015,15:1332-1347.
[34]	Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J L . Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol, 2014,32:947-951. doi: 10.1038/nbt.2969 pmid: 25038773
[35]	Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y G, Zhao K . Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One, 2016,11:e0154027. doi: 10.1371/journal.pone.0154027 pmid: 27116122
[36]	Ran F A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F . In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015,520:186-191.
[37]	Kleinstiver B P, Prew M S, Tsai S Q, Topkar V V, Nguyen N T, Zheng Z, Gonzales A P, Li Z, Peterson R T, Yeh J R, Aryee M J, Joung J K . Engineering CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 2015,523:481-485.
[38]	Hu X X, Wang C, Fu Y P, Liu Q, Jiao X Z, Wang K J . Expanding the range of CRISPR/Cas9 genome editing in rice. Mol Plant, 2016,9:943-945. doi: 10.1016/j.molp.2016.03.003 pmid: 26995294
[39]	Hu X X, Meng X B, Liu Q, Li J Y, Wang C J . Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice. Plant Biotechnol J, 2018,16:292-297. doi: 10.1111/pbi.12771 pmid: 28605576
[40]	Hiei Y, Komari T, Kubo T . Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol, 1997,35:205-218.
[41]	Wang K, Liu H Y, Du L P, Ye X G . Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties. Plant Biotechnol J, 2017,15:614-623.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

靶位点 Target site	序列 Targeted sequence (5′-3′)	GC含量 GC content (%)	外显子位置 Location
OsPDS-gRNA1	CTTGGAAGGATGAAGATGGAGA	45	3
OsPDS-gRNA2	CCAGGAGAATTCAGCCGGTTTGA	55	6
OsPDS-gRNA3	TCAGGAGAAGCATGGTTCTAAGA	45	8
OsPDS-gRNA4	TCCCGGACTGTGAACCTTGCCGA	60	13

引物名称 Primer name	序列 Sequence (5′-3′)
T7-gRNA1-FPg	TAATACGACTCACTATAGCTTGGAAGGATGAAGATGGGTTTTAGAGCTAGAAATAGC
T7-gRNA2-FPg	TAATACGACTCACTATAGCCAGGAGAATTCAGCCGGTTGTTTTAGAGCTAGAAATAGC
T7-gRNA3-FPg	TAATACGACTCACTATAGTCAGGAGAAGCATGGTTCTAGTTTTAGAGCTAGAAATAGC
T7-gRNA4-FPg	TAATACGACTCACTATAGTCCCGGACTGTGAACCTTGCGTTTTAGAGCTAGAAATAGC
gRNA-RP	AGCACCGACTCGGTGCCACTT
oligo-gRNA1-F	TTGCTTGGAAGGATGAAGATGG
oligo-gRNA1-R	AACCCATCTTCATCCTTCCAAG
oligo-gRNA2-F	TTGCCAGGAGAATTCAGCCGGTT
oligo-gRNA2-R	AACAACCGGCTGAATTCTCCTGG
oligo-gRNA4-F	TTGTCCCGGACTGTGAACCTTGC
oligo-gRNA4-R	AACGCAAGGTTCACAGTCCGGGA
PCR-gRNA1-F	TCAGAGTAAAGCAAAGATTC
PCR-gRNA1-R	TCAGGCTCCTACGAGATA
PCR-gRNA2-F	GAATTTTCGCTTAGAGGC
PCR-gRNA2-R	CCAGTTATTTGAGTTCCATC
PCR-gRNA4-F	TGTGCCTGTATGTAACCA
PCR-gRNA4-R	GAGCAAACTTCACCTTCT

名称 Name	长度 Length (bp)	G含量 G contents	C含量 C contents	GC百分比 GC percent (%)
SpCas9	4116	733	566	31.56
Cas9-codon optimized	4116	1094	931	49.20

名称 Name	阳性植株 Transplant	突变植株 Mutated plant	突变率 Mutation ratio (%)	纯合突变体 Homo plant	杂合突变体 Het plant
OsPDS-gRNA1	17/24	12	70.5	4/12	8/12
OsPDS-gRNA2	40/45	11	27.5	5/11	6/11
OsPDS-gRNA4	49/53	20	40.8	7/20	13/20