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 扩展斑马鱼和人类细胞中的 CRISPR 工具箱。


CRISPR 和 CRISPR-Cas 效应蛋白能够根据每个效应子特有的可变长度 RNA 引导，将 DNA 双链断裂靶向到定义的基因座。引导RNA的大小和形式通常相似，由与DNA靶标互补的约20个核苷酸序列和效应子识别的RNA二级结构组成。然而，效应蛋白在原型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，Cas12a家族的新成员ErCas12a在直肠真杆菌中被鉴定出来。在这里，我们报告了斑马鱼中 ErCas12a 活性的首次表征，并扩展了先前报道的人类细胞中的活性。使用荧光报告系统，我们发现 CRISPR-ErCas12a 比 CRISPR-Cas9 更有效地引发链退火介导的 DNA 修复。此外，利用我们之前报道的利用短同源性的基因打靶方法 GeneWeld，我们演示了使用 CRISPR-ErCas12a 将报告等位基因整合到斑马鱼和人类细胞的基因组中。总之，这项工作提供了部署额外 CRISPR-Cas 系统的方法，从而提高了研究人员应用基因组工程技术的灵活性。