Efficient genome editing in grapevine using CRISPR/LbCas12a system.
Efficient genome editing in grapevine using CRISPR/LbCas12a system.
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DOI：
10.1186/s43897-023-00069-w
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发表时间：
2023-10-18
影响因子：
--
通讯作者：
Liang, Zhenchang
中科院分区：
文献类型：
--
作者：
Ren, Chong;Gathunga, Elias Kirabi;Li, Xue;Li, Huayang;Kong, Junhua;Dai, Zhanwu;Liang, Zhenchang
规则间隔短回文重复序列（CRISPR）/Cas 12 a系统，也称为CRISPR/Cpf 1，已成功用于许多植物的基因组工程，但尚未在葡萄藤中应用。在这里，我们开发并证明了来自毛螺菌科细菌ND 2006（LbCas 12 a）的CRISPR/Cas 12 a在通过靶向41 B细胞中的张力性单糖转运蛋白1（TMT 1）和二氢黄酮醇-4-还原酶1（DFR 1）基因诱导靶向诱变中的功效。DFR 1基因敲除改变了DFR 1突变细胞中类黄酮的积累。热处理（34℃）提高了CRISPR/LbCas 12 a系统的编辑效率，TMT 1-crRNA 1和TMT 1-crRNA 2的编辑效率分别从35.3%提高到44.6%和29.9%提高到37.3%。此外，发现crRNA的序列是影响编辑效率的主要因素，而与设计用于多重基因组编辑的crRNA阵列内的位置无关。此外，使用截短的crRNA（trucrRNA）进行的基因组编辑显示，具有20 nt指导序列的trucrRNA与具有24 nt指导序列的原始crRNA在产生靶向诱变方面一样有效，而具有长度≤ 18 nt的较短靶互补区域的trucrRNA可能不会在41 B细胞中诱导可检测的突变。这些结果为CRISPR/LbCas 12 a系统作为基因组工程的有力工具在葡萄藤中的进一步应用提供了依据。在线版本包含补充材料，可通过10.1186/s43897-023-00069-w获得。
Clustered regularly interspaced short palindromic repeats (CRISPR) /Cas12a system, also known as CRISPR/Cpf1, has been successfully harnessed for genome engineering in many plants, but not in grapevine yet. Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1 (TMT1) and dihydroflavonol-4-reductase 1 (DFR1) genes in 41B cells. Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells. Heat treatment (34℃) improved the editing efficiencies of CRISPR/LbCas12a system, and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3% to 44.6% and 29.9% to 37.3% after heat treatment, respectively. Moreover, the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing. In addition, genome editing with truncated crRNAs (trucrRNAs) showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis, whereas trucrRNAs with shorter regions of target complementarity ≤ 18 nt in length may not induce detectable mutations in 41B cells. All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering. The online version contains supplementary material available at 10.1186/s43897-023-00069-w.
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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
DOI：
10.1126/science.abb1400
发表时间：
2020-07-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Pausch P;Al-Shayeb B;Bisom-Rapp E;Tsuchida CA;Li Z;Cress BF;Knott GJ;Jacobsen SE;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
13.8
作者：
Li B;Liang S;Alariqi M;Wang F;Wang G;Wang Q;Xu Z;Yu L;Naeem Zafar M;Sun L;Si H;Yuan D;Guo W;Wang Y;Lindsey K;Zhang X;Jin S
通讯作者：
Jin S
影响因子：
46.9
作者：
通讯作者：
--
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