Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing
Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing
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DOI：
10.1038/s41587-018-0011-0
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发表时间：
2019-03-01
影响因子：
46.9
通讯作者：
Joung, J. Keith
中科院分区：
文献类型：
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作者：
Kleinstiver, Benjamin P.;Sousa, Alexander A.;Joung, J. Keith
CRISPR-Cas 12 a（以前的Cpf 1）核酸酶（1）的广泛使用受到对扩展的TTTV原型间隔区邻近基序（PAM）的需求的阻碍（2）。为了解决这一限制，我们设计了一种增强的氨基酸球菌Cas 12 a变体（enAsCas 12 a），其具有大幅扩展的靶向范围，能够靶向许多以前无法获得的PAM。平均而言，与野生型AsCas 12 a相比，enAsCas 12 a在具有典型TTTV PAM的位点上表现出高出两倍的基因组编辑活性，并且我们成功地将enAsCas 12 a的突变子集移植到其他先前描述的AsCas 12 a变体上（3）以增强其活性。enAsCas 12 a提高了多重基因编辑、内源性基因激活和C-to-T碱基编辑的效率，我们设计了一种高保真版本的enAsCas 12 a（enAsCas 12 a-HF 1）以减少脱靶效应。当作为核糖核蛋白（RNP）复合物递送时，enAsCas 12 a和enAsCas 12 a-HF 1两者在HEK 293 T和原代人T细胞中起作用。总的来说，enAsCas 12 a提供了Cas 12 a的优化版本，这应该能够使Cas 12 a酶更广泛地应用于基因和表观遗传编辑。
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases(1) has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)(2). To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants(3) to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.