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Abstract：
The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.
摘要：
RNA指导的AsCas12a核酸酶的效率。与化脓性链球菌的SpCas9进行了比较，用于曼氏血吸虫的功能基因组学。我们对引导RNA与核酸酶的比例进行了优化条件，捐赠者模板，和电穿孔参数，靶向一种称为omega-1的关键血吸虫酶。Cas12a和Cas9催化的程序分裂导致交错的和钝端链断裂，分别。AsCas12a在基因敲除方面比SpCas9更有效，通过潮流分析确定。CRISPResso2分析证实大多数突变是缺失。在存在单链寡脱氧核苷酸（ssODN）模板的情况下，两种核酸酶的敲除效率均显着增加。有了AsCas12a，测试了代表非CRISPR靶标（NT）和靶标（T）链的ssODN，在SpCas9加ssODN中，KO效率分别为15.67、28.71和21.43%，AsCas12a加NT-ssODN，和AsCas12a加上T-ssODN组，分别。活化的AsCas12a对ssODN的反式切割在体外不明显。SpCas9催化更精确的转基因插入，KI_Cas9组的敲入效率为17.07%，KI_Cas12a-NT-ssODN占14.58%，KI_Cas12a-T-ssODN为12.37%。尽管与SpCas9相比，AsCas12a在每个基因组中诱导的突变较少，但两种核酸酶对omega-1转录和表达的表型影响相似。
