High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system.
High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system.
复制标题
使用CRISPR/CAS9系统中的同种异体纤维棉（Gossypium hirsutum）中的高效多源基因组编辑。
DOI：
10.1111/pbi.12755
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发表时间：
2018-01
影响因子：
13.8
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Wang P;Zhang J;Sun L;Ma Y;Xu J;Liang S;Deng J;Tan J;Zhang Q;Tu L;Daniell H;Jin S;Zhang X
陆地棉是具有复杂基因组的异源四倍体。大多数基因具有属于At和Dt亚基因组的多个拷贝。基因同源物之间的序列相似性也非常高。为了有效地实现位点/基因特异性突变是非常必要的。由于其高效性和鲁棒性，CRISPR（成簇的规则间隔短回文重复序列）/Cas9系统已经从原核生物到真核生物发挥了广泛的位点特异性基因组编辑。在这项研究中，我们利用CRISPR/Cas9系统在单个载体中产生两个sgRNA，以在异源四倍体棉花中进行多位点基因组编辑。选择外源转化基因Discosoma红色荧光蛋白2（DsRed 2）和内源基因GhCLA 1作为靶基因。T0代的DsRed 2编辑植物将其性状恢复为野生型，整个植物的红色荧光消失。此外，突变的表型和基因型遗传到它们的T1后代。对于内源基因GhCLA 1，75%的再生植株表现为白化病表型，并有明显的核苷酸和DNA片段缺失。每个靶位点的基因编辑效率为66.7- 100%。用桑格测序检查两种基因的突变基因型。在GhCLA 1编辑的T0植物中进行了基于条形码的高通量测序，该测序可高效地对突变体群体进行基因分型，并且与桑格测序结果匹配良好。在潜在脱靶部位未检测到脱靶编辑。这些结果证明CRISPR/Cas9系统对于异源四倍体棉花基因组编辑是高效和可靠的。
Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.
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影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
第一代杨树中 CRISPR/Cas9 介导的高效定点诱变
影响因子：
4.6
作者：
Fan D;Liu T;Li C;Jiao B;Li S;Hou Y;Luo K
通讯作者：
Luo K
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle