CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

中文翻译：

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使用ErCas12a在Zebrafish和人类细胞中扩展CRISPR Toolbox。

CRISPR和CRISPR-Cas效应子蛋白可根据特定于每个效应子的可变长度RNA向导，将DNA双链断裂靶向到定义的基因座。指导RNA的大小和形式通常相似，由与DNA靶标互补的〜20个核苷酸序列和效应子识别的RNA二级结构组成。但是，效应蛋白在原间隔子相邻基序要求，核酸酶活性和DNA结合动力学方面有所不同。最近，在真细菌中发现了Cas12a家族的新成员ErCas12a。在这里，我们报告了斑马鱼中ErCas12a活性的第一个特征，并扩展了人类细胞中先前报道的活性。使用荧光报告系统，我们显示CRISPR-ErCas12a比CRISPR-Cas9更有效地引发链退火介导的DNA修复。此外，使用我们先前报道的利用短同源性的基因靶向方法GeneWeld，我们证明了CRISPR-ErCas12a将报道等位基因整合到斑马鱼和人类细胞的基因组中的用途。总之，这项工作为部署额外的CRISPR-Cas系统提供了方法，从而提高了研究人员在应用基因组工程技术中的灵活性。