Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.
Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.
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DOI：
10.1038/s41587-021-01009-z
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发表时间：
2022-01
影响因子：
46.9
通讯作者：
Kim YS
中科院分区：
文献类型：
--
作者：
Kim DY;Lee JM;Moon SB;Chin HJ;Park S;Lim Y;Kim D;Koo T;Ko JH;Kim YS
基因治疗将受益于微型CRISPR系统，该系统适合小型腺相关病毒（AAV）基因组，并在真核细胞中具有高切割活性和特异性。迄今为止发现的最紧凑的CRISPR相关核酸酶之一是古细菌Un 1Cas 12 f1。然而，Un 1Cas 12 f1及其变体在真核细胞中具有非常低的活性。在本研究中，我们在5个位点重新设计了Un 1Cas 12 f1的天然指导RNA：反式激活CRISPR RNA（tracrRNA）的5′末端，tracrRNA-crRNA互补区，五（尿苷酸）序列，crRNA的3′末端和tracrRNA中的无序茎2区。这些优化协同地将平均插入缺失频率提高了867倍。当通过质粒载体、PCR扩增子和AAV递送时，优化的Un 1Cas 12 f1系统能够在人类细胞中进行高效、特异性的基因组编辑。由于Un 1Cas 12 f1在原型间隔区之外切割，因此它可以用于有效地产生大的缺失。工程化的Un 1Cas 12 f1系统显示出与SpCas 9相当的效率和与AsCas 12 a相似的特异性。微型CRISPR系统可以使用单个AAV载体实现基因组编辑。
Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a. A miniature CRISPR system may enable genome editing using single AAV vectors.
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DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harrington LB;Burstein D;Chen JS;Paez-Espino D;Ma E;Witte IP;Cofsky JC;Kyrpides NC;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
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：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle