Journal: bioRxiv
Article Title: ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT A SCHISTOSOMA MANSONI GENOME SAFE HARBOR
doi: 10.1101/2023.09.12.557428
Figure Lengend Snippet: Editing GSH1 in S. mansoni showed an efficiency bias for Cas12a vs. Cas9 and prominent deletion patterns at this target locus. Synopsis of the comparative approach using Cas9 and Cas12a for programmed gene editing with the S. mansoni egg as biological target for transformation by electroporation. (A) Shown is a schematic overview of the GSH1 (SM_V7_Chr3; 13380432-13381848; 1,416 bp) target sites for Cas9 (blue arrow) and Cas12a (red arrow). DNA cleavage by Cas9 was expected to result in a blunt-ended cut, whereas Cas12a digested released overhanging DNA strands. The non-target DNA strand at GSH1was expected to be cleaved at the same position as illustrated. GSH1 regions highlighted in magenta represent the short homology arms, 50 nt used for the donor template. (B) LEs were subjected to electroporation in the presence of CX Rhodamine-labelled RNPs (red), after which they were counterstained with Hoechst33342 (blue) and examined by CLSM 24 h later. Introduction of RNPs was demonstrated (arrows) with both nucleases. The majority of Cas9 RNPs accumulated around the eggshell, a dissimilar pattern to that observed in the Cas12a RNP-treated LEs, where signals were also found inside the eggs. (C) Results of the determination of editing efficiencies by InDel abundance generated by Cas9 and Cas12a RNPs in GSH1, as analyzed by TIDE analysis . The editing efficiencies of GSH1-targeting RNPs normalized to the wild-type sequence (KO-Cas9, dark blue bar; KO-Cas12a, red bar) and RNPs non-specific to the S. mansoni genome (scramble-Cas9, purple bar; scramble-Cas12a, orange bar), respectively are shown. The editing efficiency of LEs treated with RNPs were significantly higher compared to LEs from the scramble control (*P < 0.05, **P < 0.01, ***P < 0.001; t test, n = 9). Compared with Cas9-RNPs, treatment with Cas12a-RNPs (red) showed a statistically significant higher editing efficiency. (D) , Representative examples of NGS reads or Cas12a and Cas9, respectively, to show alleles with frequently seen profiles of deletions at the programmed cleavage positions e (red arrows) (n = 3). Abbreviations: CLSM, confocal laser scanning microscopy; KO, knock-out, ns, not statistic significant; wt, wild type.
Article Snippet: Generated amplicons were suitable for the AmEZ service for next generation sequencing (NGS) (Azenta Life Sciences).
Techniques: Transformation Assay, Electroporation, Generated, Sequencing, Confocal Laser Scanning Microscopy, Knock-Out
