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Question

a Cells targeted with 8 µg G5 sgRNA/15 µg Cas9 RNP and 5.4 µg of 72-mer ssODN and analyzed with TIDER software for percent indels and HDR. This concentration of CRISPR/Cas9 and ssODN was found to be the optimal concentrations, in our hands, for targeting the CD34+ cells with significant HDR. The graph represents data from two experiments targeting the same source of CD34+ cells with CRISPR/Cas9 with the G5 sgRNA (#1 and #2 are independent experiments), and two experiments targeting the same CD34+ cells with CRIPSR/Cas9 with the G10 sgRNA (#1 and #2 for each experimental set). b, c Representative TIDER plots of indel and HDR activity catalyzed by the G5 sgRNA and G10 sgRNA CRISPR/Cas9 (experimental set #1 for each CRISPR/Cas9). The majority of the indels are 5 bp deletion or less when using CRISPR/Cas9 close to the mutation site, as can be seen by the black box (b), while there are larger sized indels when using the CRISPR/Cas9 that cuts further away from the HBB mutation site. The data presented in this figure are from patient 8 CD34+ cells that were targeted using both the G5 and G10 CRISPR/Cas9 RNPs and 72-mer ssODN, in duplicate reactions.

Answer 1

Optimal concentrations of CRISPR/Cas9 and ssODN were found to result in significant HDR in targeted CD34+ cells, with TIDER software analysis showing mostly 5 bp or less indels near the mutation site when using the G5 sgRNA.