Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.
Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.
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DOI：
10.1093/plphys/kiac128
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发表时间：
2022-06-01
期刊：
影响因子：
7.4
通讯作者：
Tsai, Chung-Jui
中科院分区：
文献类型：
--
作者：
Bewg, W. Patrick;Harding, Scott A.;Engle, Nancy L.;Vaidya, Brajesh N.;Zhou, Ran;Reeves, Jacob;Horn, Thomas W.;Joshee, Nirmal;Jenkins, Jerry W.;Shu, Shengqiang;Barry, Kerrie W.;Yoshinaga, Yuko;Grimwood, Jane;Schmitz, Robert J.;Schmutz, Jeremy;Tschaplinski, Timothy J.;Tsai, Chung-Jui
随着CRISPR/Cas编辑植物的重点从概念验证转移到现实世界的应用，精确的基因操作将越来越需要对多基因性状进行并发的多重编辑。跨多个位点编辑的常见方法是为每个靶标设计一个指导RNA（gRNA）;然而，这使构建体组装复杂化并增加了脱靶突变的可能性。在这项研究中，我们利用一种gRNA靶向MYB 186（一种已知的正毛状体调节剂）及其旁系同源物MYB 138和MYB 38在杂交白杨（美洲山杨×银白杨INRA 717- 1B 4）中的共有位点进行诱变。MYB 186和MYB 138的意外重复导致了杂种白杨中3个靶基因的8个等位基因。深度测序和聚合酶链反应分析证实了几乎所有产生的无毛突变体中的所有八个靶标的编辑，范围从小的indel到大的基因组缺失，在四个潜在位点没有检测到脱靶活性。这突出了靶向保守外显子区域的单个gRNA用于多重编辑的有效性。此外，角质层蜡和全叶分析表明，在无毛突变体中完全不存在三萜，暗示了以前未描述的作用，为白杨的非腺毛。 用单个gRNA靶向保守序列允许多基因家族的有效诱变以及无毛和无三萜的白杨突变体的恢复。
As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar. Targeting conserved sequences with a single gRNA allows efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.
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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：
10.1007/bf00331014
发表时间：
1986-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
KONCZ, C;SCHELL, J
通讯作者：
SCHELL, J
影响因子：
27.5
作者：
Ma, Xingliang;Zhang, Qunyu;Liu, Yao-Guang
通讯作者：
Liu, Yao-Guang
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
用于多重植物基因组编辑和转录调控的 CRISPR/Cas9 工具箱
影响因子：
7.4
作者：
Lowder, Levi G.;Zhang, Dengwei;Qi, Yiping
通讯作者：
Qi, Yiping