Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems.
Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems.
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利用 Cas12a 直向同源物和高度多路复用的编辑系统扩大植物基因组工程的范围
DOI：
10.1038/s41467-021-22330-w
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发表时间：
2021-03-29
影响因子：
16.6
通讯作者：
Qi Y
中科院分区：
文献类型：
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作者：
Zhang Y;Ren Q;Tang X;Liu S;Malzahn AA;Zhou J;Wang J;Yin D;Pan C;Yuan M;Huang L;Yang H;Zhao Y;Fang Q;Zheng X;Tian L;Cheng Y;Le Y;McCoy B;Franklin L;Selengut JD;Mount SM;Que Q;Zhang Y;Qi Y
CRISPR-Cas 12 a是一种很有前途的基因组编辑系统，用于靶向富含AT的基因组区域。全面的基因组工程需要同时靶向多个基因在定义的位置。为了扩大Cas 12 a的靶向范围，我们筛选了9个尚未在植物中证实的Cas 12 a直向同源物，并鉴定了6个在水稻中具有高编辑活性的同源物，ErCas 12 a，Lb 5Cas 12 a，BsCas 12 a，Mb 2Cas 12 a，TsCas 12 a和MbCas 12 a。其中，Mb 2Cas 12 a以高编辑效率和耐低温性脱颖而出。工程改造的Mb 2Cas 12 a-RVRR变体能够在水稻中以更宽松的PAM要求进行编辑，产生比野生型SpCas 9高两倍的基因组覆盖率。为了实现大规模基因组工程，我们比较了12个多重Cas 12 a系统，并确定了一个有效的系统，该系统表现出近100%的双等位基因编辑效率，能够靶向水稻中多达16个位点。这是迄今为止使用Cas 12 a在植物中进行的最高水平的多重编辑。我们还开发了两个紧凑的单转录单位CRISPR-Cas 12 a干扰系统，用于水稻和拟南芥中的多基因阻遏。该研究极大地扩展了Cas 12 a用于作物基因组工程的靶向范围。
CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice andArabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.