Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

Chao Feng; Jing Yuan; Rui Wang; Yang Liu; James A. Birchler; Fangpu Han

doi:10.1016/j.jgg.2015.10.002

Volume 43 Issue 1

Jan. 2016

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2016 > 43(1): 37-43

PDF( 1412 KB)

Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System

doi: 10.1016/j.jgg.2015.10.002

Chao Feng^{a, b, #},
Jing Yuan^{a, #},
Rui Wang^a,
Yang Liu^{a, b},
James A. Birchler^c,
Fangpu Han^a

a
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA

More Information

Corresponding author: E-mail address: fphan@genetics.ac.cn (Fangpu Han)
Received Date: 2015-08-16
Revised Date: 2015-10-14
Accepted Date: 2015-10-20

Available Online: 2015-10-30

Publish Date: 2016-01-20

Abstract

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker geneZmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.
- CRISPR/Cas9,
- Targeted genome modification,
- Heterochromatic region,
- Maize

These authors contributed equally to this work.

FullText(HTML)

References(38)

References

[1]	Bassett, A.R., Liu, J.L. J. Genet. Genomics, 41 (2014),pp. 7-19
[2]	Candela, H., Hake, S. The art and design of genetic screens: maize Nat. Rev. Genet., 9 (2008),pp. 192-203
[3]	Carroll, D. Genome engineering with zinc-finger nucleases Genetics, 188 (2011),pp. 773-782
[4]	Cho, S.W., Kim, S., Kim, J.M. et al. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease Nat. Biotechnol., 31 (2013),pp. 230-232
[5]	Cong, L., Ran, F.A., Cox, D. et al. Multiplex genome engineering using CRISPR/Cas systems Science, 339 (2013),pp. 819-823
[6]	Dillon, N., Festenstein, R. Unravelling heterochromatin: competition between positive and negative factors regulates accessibility Trends Genet., 18 (2002),pp. 252-258
[7]	Feng, Z.Y., Mao, Y., Xu, N. et al. Proc. Natl. Acad. Sci. USA, 111 (2014),pp. 4632-4637
[8]	Feng, Z.Y., Zhang, B.T., Ding, W.N. et al. Efficient genome editing in plants using a CRISPR/Cas system Cell Res., 23 (2013),pp. 1229-1232
[9]	Fauser, F., Schiml, S., Puchta, H. Plant J., 79 (2014),pp. 348-359
[10]	Frame, B.R., Shou, H., Chikwamba, R.K. et al. Plant Physiol., 129 (2002),pp. 13-22
[11]	Fu, S., Lv, Z., Gao, Z. et al. Proc. Natl. Acad. Sci. USA, 110 (2013),pp. 6033-6036
[12]	Fu, Y., Foden, J.A., Khayter, C. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Nat. Biotechnol., 31 (2013),pp. 822-826
[13]	He, G., Chen, B., Wang, X. et al. Conservation and divergence of transcriptomic and epigenomic variation in maize hybrids Genome Biol., 14 (2013),p. R57
[14]	Hsu, P.D., Lander, E.S., Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering Cell, 157 (2014),pp. 1262-1278
[15]	Hwang, W.Y., Fu, Y.F., Reyon, D. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system Nat. Biotechnol., 31 (2013),pp. 227-229
[16]	Jia, H., Wang, N. Targeted genome editing of sweet orange using Cas9/sgRNA PLoS One, 9 (2014),p. e93806
[17]	Jiang, W.Y., Bikard, D., Cox, D. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems Nat. Biotechnol., 31 (2013),pp. 233-239
[18]	Jinek, M., Chylinski, K., Fonfara, I. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science, 337 (2012),pp. 816-821
[19]	Johnson, R.A., Gurevich, V., Filler, S. et al. Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta Plant Mol. Biol., 87 (2015),pp. 143-156
[20]	Joung, J.K., Sander, J.D. TALENs: a widely applicable technology for targeted genome editing Nat. Rev. Mol. Cell Biol., 14 (2012),pp. 49-55
[21]	Leader, D.J., Connelly, S., Filipowicz, W. et al. Biochim. Biophys. Acta, 1219 (1994),pp. 145-147
[22]	Li, D.L., Qiu, Z.W., Shao, Y.J. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system Nat. Biotechnol., 31 (2013),pp. 681-683
[23]	Li, J.F., Norville, J.E., Aach, J. et al. Nat. Biotechnol., 31 (2013),pp. 688-691
[24]	Liang, Z., Zhang, K., Chen, K. et al. J. Genet. Genomics, 41 (2014),pp. 63-68
[25]	Lu, X.M., Hu, X.J., Zhao, Y.Z. et al. Mol. Plant, 5 (2012),pp. 1100-1112
[26]	Mali, P., Yang, L., Esvelt, K.M. et al. Science, 339 (2013),pp. 823-826
[27]	Nekrasov, V., Staskawicz, B., Weigel, D. et al. Nat. Biotechnol., 31 (2013),pp. 691-693
[28]	Pattanayak, V., Lin, S., Guilinger, J.P. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity Nat. Biotechnol., 31 (2013),pp. 839-843
[29]	Perez-Pinera, P., Ousterout, D.G., Gersbach, C.A. Advances in targeted genome editing Curr. Opin. Chem. Biol., 16 (2012),pp. 268-277
[30]	Puchta, H., Fauser, F. Gene targeting in plants: 25 years later Int. J. Dev. Biol., 57 (2013),pp. 629-637
[31]	Shan, Q.W., Wang, Y.P., Li, J. et al. Targeted genome modification of crop plants using a CRISPR-Cas system Nat. Biotechnol., 31 (2013),pp. 686-688
[32]	Svitashev, S., Young, J., Schwartz, C. et al. Targeted mutagenesis, precise gene editing and site-specific gene insertion in maize using Cas9 and guide RNA Plant Physiol., 169 (2015),pp. 931-945
[33]	Wolfgruber, T.K., Sharma, A., Schneider, K.L. et al. Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic loci shaped primarily by retrotransposons PLoS Genet., 5 (2009),p. e1000743
[34]	Xing, H.L., Dong, L., Wang, Z.P. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants BMC Plant Biol., 14 (2014),p. 327
[35]	Xu, R., Li, H., Qin, R. et al. Rice, 7 (2014),p. 5
[36]	Yu, Z., Ren, M., Wang, Z. et al. Genetics, 195 (2013),pp. 289-291
[37]	Zhang, H., Zhang, J., Wei, P. et al. The CRISPR/Cas9 system produces specific and homozygous targete gene editing in rice in one gerneration Plant Biotech. J., 12 (2014),pp. 797-807
[38]	Zhang, Y., Su, J., Duan, S. et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes Plant Methods, 7 (2011),p. 30