Fig. 3: On-target integration events in G418-resistant clones by rAAV and rAAV-CRISPR/Cas9.

a PCR-based identification of on-target events in SF3B1 alleles using rAAV-based homologous recombination alone strategy. Four of the 25 (16%) G418-resistant clones were PCR positive for on-target integration of the expression cassette (marked with asterisks); C control wild type. b Depicted is the Sanger sequencing of the gDNA target sequence of one of the representative PCR-positive clones generated by rAAV-alone-based HR. The clone did not undergo the desired K700E modification indicating failure of HR between the target DNA and rAAV in region of the desired modification (∼300 bp from the gene-trap cassette integration). c Illustration of the HR event between rAAV vector and genome locus. In the absence of a targeted DSB, HR begins in the intron at the site of obligatory Neo^R intron-trap insertion (depicted in yellow) spreading out with decreasing efficiency before falling off due to significant homology between the rAAV HAs and the host genome. d PCR-based identification of on-target events in SF3B1 alleles using rAAV + CRISPR/Cas9-based homologous recombination strategy. Thirty-one of the 33 (94%) G418-resistant clones demonstrated on-target integration of the expression cassette (marked with asterisks). e Depicted is the Sanger sequencing of the gDNA target sequence of one of the representative PCR-positive clones generated by rAAV-CRISPR/Cas9-based HR, showing successful single-allele editing of the SF3B1 K700 locus. f The double-stranded break introduced by Cas9 facilitates locus-specific HR of the nascent DNA strand with the rAAV donor template containing the desired mutation.