RNA-guidedAsCas12a- andSpCas9-catalyzed knockout and homology directed repair of theomega-1locus of the human blood fluke,Schistosoma mansoni

We compared the efficiency of gene knockout (KO) and precision of insertion (knock-in, KI) of the RNA-guided As Cas12a nuclease of Acidaminococcus sp. with that of Sp Cas9 from Streptococcus pyogenes , aiming to enhance the functional genomics toolkit for Schistosoma mansoni . Programmed DNA cleavages catalyzed by Cas12a and Cas9 result in staggered and blunt ended strand breaks, respectively. TTTV, the optimal protospacer adjacent motif for As Cas12a would occur frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key enzyme termed omega-1 that is secreted by the schistosome egg. As Cas12a was more efficient than Sp Cas9 for gene knockout of omega-1 as determined by tracking of indels by decomposition ( P < 0.001). Resulting from CRISPREsso2 analysis, most mutations were deletions; Sp Cas9 induced short deletions of 3 nt in length whereas As Cas12a induced deletions of 2 to 26 nt. Knockout efficiency of both nucleases markedly increased in the presence of short, single stranded oligodeoxynucleotide (ssODN) donor templates. With As Cas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands of the targeted gene were tested, resulting in KO efficiencies of 15.67, 28.71 and 21.43% in the Sp Cas9 plus donor ssODN, As Cas12a plus NT-ssODN, and As Cas12a plus T-ssODN groups, respectively. Trans cleavage activity against the ssODNs by activated As Cas12a was not apparent in vitro . Programmed Sp Cas9 editing led to more precise transgene insertion than As Cas12a, with KI efficiencies of 17.07% for the KI_SpCas9 group, 14.58% for KI_ As Cas12a-NT-ssODN and 12.37% for KI_ As Cas12a-T-ssODN. Although As Cas12a induced fewer mutations per genome than Sp Cas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. These findings revealed that As Cas12a and Sp Cas9 both provide tractable routes for RNA-guided programmed mutation of the genome of the schistosome egg.