Genome-Wide Assessment of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis

被引：107

作者：

Peterson, Brenda A. ^{[1
,5
]}

Haak, David C. ^{[6
]}

Nishimura, Marc T. ^{[4
]}

Teixeira, Paulo J. P. L. ^{[4
]}

James, Sean R. ^{[1
,5
]}

Dangl, Jeffery L. ^{[1
,2
,3
,4
]}

Nimchuk, Zachary L. ^{[1
,2
,5
]}

机构：

[1] Univ North Carolina Chapel Hill, Dept Biol, Chapel Hill, NC 27514 USA

[2] Univ North Carolina Chapel Hill, Carolina Ctr Genome Sci, Curriculum Genet & Mol Biol, Chapel Hill, NC 27514 USA

[3] Univ North Carolina Chapel Hill, Dept Microbiol & Immunol, Chapel Hill, NC USA

[4] Univ North Carolina Chapel Hill, Howard Hughes Med Inst, Chapel Hill, NC USA

[5] Virginia Tech, Dept Biol Sci, Blacksburg, VA 24061 USA

[6] Virginia Tech, Dept Plant Pathol Physiol & Weed Sci, Blacksburg, VA USA

来源：

PLOS ONE | 2016年 / 11卷 / 09期

基金：

美国国家科学基金会;

关键词：

CHROMOSOMAL TRANSLOCATIONS; RNA; MUTAGENESIS; CAS9; VISUALIZATION; PEPTIDES; TALENS;

D O I：

10.1371/journal.pone.0162169

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate thatmultiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for cleanmutant generation with no need to enhance specificity or adopt alternate Cas9 variants.

引用

页数：11

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