Fig. 2: Genome editing by Cas12b nucleases in human 293FT cells.
From: Repurposing CRISPR-Cas12b for mammalian genome engineering
a Schematic illustration of the eukaryotic expression strategy of the Cas12b and its cognate guide RNA (gRNA). The pre-tRNAGly, an endogenous RNA processing system for cleavage of transcripts, was hijacked to simultaneously express the tracrRNA and crRNA using a single human U6 promoter. b Schematic illustration of the human RNF2 target site 1 and crRNA/tracrRNA duplex. Red letters indicate the PAM sequences. c T7EI analysis of the indels produced by Cas12b orthologs (AaCas12b, AkCas12b, AmCas12b, and BsCas12b) at the human RNF2 target site 1. The indel rate is shown under the lanes with mutation. mock, an U6 empty vector without crRNA/tracrRNA expression. GFP, an empty backbone vector without Cas12b protein expression. d Sanger sequencing results showing the indels in human RNF2 target site 1 produced by AaCas12b and AkCas12b. Blue dashes, deleted bases; purple lowercases, insertions or mutations; red uppercases, PAM. e Effects of human plasma incubation on the nuclease activity of SpCas9 and AaCas12b. After incubation in human plasma at indicated concentrations for 12 h at 37 °C, in vitro DNA cleavage assay was conducted. The cleavage rate is shown under the cleaved lanes
