Fig. 5: SECRETS is effective at identifying highly specific x-gRNAs for clinically relevant gRNAs.
From: Selection of extended CRISPR RNAs with enhanced targeting and specificity
a In vitro validation of 3 x-gRNAs for a gRNA used in a treatment of sickle-cell disease (SCD) targeting the HBB gene from ClinicalTrials.gov ID NCT04774536 (see main text). While the regular gRNA exhibits significant off-target activity, x-gRNAs identified via SECRETS were able to completely abolish detectable off-target activity while maintaining activity on-target comparable to the standard gRNA. b A workflow for how SECRETS might improve the safety in the protocols for developing personalized gene therapies. Once an individual has been recognized as a candidate for gene therapy and a target for Cas9 determined, the target is used to determine a high-efficiency gRNA sequence to be used for therapy and itself cloned into the plasmid with a toxin (pSECRETS-C). The gRNA is then used to screen for sequences where off-target activity might occur specifically in that individual’s genome (e.g. using CHANGE-Seq5) and cloned into the x-gRNA library plasmid with a kanamycin resistance cassette (pSECRETS-B) along with the off-target sequence(s) to avoid. After SECRETS, sequencing, and in vitro validation, highly specific x-gRNAs for that personalized gene therapy will have been efficiently generated.
