Fig. 5: HDR-boosting modules function in other types of precise gene editing.
From: Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor
a Schematic representation for DNA knock-in at the endogenous genomic loci cooperating with Cas9. b FLAG or loxP sequence insertion efficiency mediated by the indicated ssDNA donors at the FANCF gene locus in HeLa cells. 18 pmol Cas9 nuclease, 22 pmol gRNA and ssDNA donors with the indicated amount (per million cells) corresponded to 2 × 105 cells. c Schematic illustrating DNA double nicks using a pair of sgRNAs guiding Cas9 nickases (nCas9). The D10A mutation renders Cas9 able to cleave only the strand complementary to the sgRNA; the H840A mutation renders Cas9 able to cleave only the non-complementary strand. A pair of sgRNA-nCas9 complexes can nick both strands simultaneously. d HDR efficiencies in paired nCas9-mediated precise editing system using ssDNA donors tethered with HDR-boosting module. In this electroporation, 36 pmol nCas9 nuclease, 22 pmol gRNA, 22 pmol AS-gRNA and the indicated amount of ssDNA donor (per million cells) corresponded to 2 × 105 cells. e Schematic representation for gene editing using the Cas12a nuclease which generates sticky-end DSB. f HDR efficiencies of HDR-boosting modular ssDNA donors and canonical ssDNA donors cooperated with Cas12a at the RNF2 site in HEK 293T cells. 32 pmol Cas12a nuclease, 37.5 pmol gRNA and 6 pmol ssDNA donors corresponded to 2 × 105 cells. For all HDR efficiency-assessing experiments, HDR efficiency was measured three days after electroporation. Values and error bars reflect mean ± SD of n = 3 (d) independent electroporation replicates. Values reflect n = 2 (b, f) independent electroporation replicates. The sequences of all gRNAs and ssDNA donors used are shown in Supplementary Data 1 and 2. Source data are provided as a Source Data file. Figure (a, c, e) Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
