Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize.
Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize.
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DOI：
10.1111/pbi.12982
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发表时间：
2019-03
影响因子：
13.8
通讯作者：
Wang K
中科院分区：
文献类型：
--
作者：
Lee K;Zhang Y;Kleinstiver BP;Guo JA;Aryee MJ;Miller J;Malzahn A;Zarecor S;Lawrence-Dill CJ;Joung JK;Qi Y;Wang K
CRISPR/Cas9和Cas12a （Cpf1）核酸酶是植物中两种最强大的基因组编辑工具。在这项工作中，我们通过针对具有重叠序列的玉米glossy2基因编码区来比较它们的活性。我们通过农杆菌介导的转化，将携带SpCas9‐引导RNA （gRNA）和LbCas12a‐CRISPR RNA （crRNA）的构建体导入玉米自交系B104胚胎。靶突变分析显示，90%-100%的Cas9编辑的T0植株携带indel突变，63%-77%为纯合或双等位突变。相比之下，0%-60%的Cas12a编辑的T0植物具有靶突变。然后，我们进行了CIRCLE - seq分析，以确定全基因组范围内Cas9的潜在脱靶位点。两种grna共鉴定出18个和67个潜在的脱靶位点，与靶位点相比平均有5个错配。对部分脱靶位点的测序分析显示，T1植株中没有可检测到的突变水平，它们组成性地表达Cas9核酸酶和grna。综上所述，我们的研究结果表明，本研究使用的CRISPR/Cas9系统对玉米基因组编辑具有高效和特异性，而CRISPR/Cas12a系统需要进一步优化以提高编辑效率。
CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.
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影响因子：
4.6
作者：
Endo A;Masafumi M;Kaya H;Toki S
通讯作者：
Toki S
影响因子：
14.9
作者：
Andorf CM;Cannon EK;Portwood JL 2nd;Gardiner JM;Harper LC;Schaeffer ML;Braun BL;Campbell DA;Vinnakota AG;Sribalusu VV;Huerta M;Cho KT;Wimalanathan K;Richter JD;Mauch ED;Rao BS;Birkett SM;Sen TZ;Lawrence-Dill CJ
通讯作者：
Lawrence-Dill CJ
影响因子：
7.3
作者：
Housden BE;Valvezan AJ;Kelley C;Sopko R;Hu Y;Roesel C;Lin S;Buckner M;Tao R;Yilmazel B;Mohr SE;Manning BD;Perrimon N
通讯作者：
Perrimon N
影响因子：
46.9
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
通讯作者：
Joung JK
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F