Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.
Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.
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通过 Cas12a 变体介导的胞嘧啶和腺嘌呤碱基编辑器进行多重碱基编辑
DOI：
10.1038/s42003-022-04152-8
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发表时间：
2022-11-02
影响因子：
5.9
通讯作者：
中科院分区：
文献类型：
--
作者：
Cas12a可以处理来自CRISPR阵列的单个转录物的多个sgRNA，当掺入碱基编辑器系统时，赋予多重碱基编辑的优势，这是非常有帮助的，因为表型通常涉及多个基因或单核苷酸变体。然而，通过Cas12a衍生的碱基编辑器进行的多重碱基编辑几乎没有报道，主要是由于野生型Cas12a的TTTV的效率受损和受限制的原间隔区邻近基序（PAM）。在这里，我们开发了Cas12a介导的胞嘧啶碱基编辑器（CBE）和腺嘌呤碱基编辑器（ABE）系统，通过将高活性脱氨酶与毛螺菌科细菌Cas12a（LbCas12a）变体相结合，提高了效率并扩大了靶向范围。我们证实，这些CBE和ABE可以执行有效的C到T和A到G的转换，分别与PAM的NTTN，TYCN，和TRTN的目标。值得注意的是，可以在体细胞和胚胎中使用开发的CBE和ABE进行多重碱基编辑。这些Cas12a变体介导的碱基编辑器将作为多重点突变的通用工具，这在遗传改良、疾病建模和基因治疗中非常重要。通过将高活性脱氨酶与识别非TTTV PAM的Cas12a变体enCas12a、RR和RVR组合，开发了具有提高的效率和扩展的靶范围的Cas12a介导的胞嘧啶（CBE）和腺嘌呤碱基编辑器（ABE）系统。
Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy. Cas12a-mediated cytosine (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded target scopes are developed, by combining highly active deaminases with Cas12a variants, enCas12a, RR and RVR, recognizing non-TTTV PAMs.
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影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
DOI：
10.1007/s42994-020-00018-x
发表时间：
2020-04-01
期刊：
ABIOTECH
影响因子：
--
作者：
Molla, Kutubuddin A.;Shih, Justin;Yang, Yinong
通讯作者：
Yang, Yinong
影响因子：
48
作者：
Breinig, Marco;Schweitzer, Anabel Y.;Tschaharganeh, Darjus F.
通讯作者：
Tschaharganeh, Darjus F.
影响因子：
46.9
作者：
Gaudelli, Nicole M.;Lam, Dieter K.;Ciaramella, Giuseppe
通讯作者：
Ciaramella, Giuseppe
影响因子：
--
作者：
Brunet E;Jasin M
通讯作者：
Jasin M