Shuttle peptides deliver Cas12a and Cas9 RNPs to HAE. A) Schematic showing CFTR locus in region of 3849 + 10C>T mutation (not to scale) and the sequence of the Cas12a guide RNA target. B) Editing at the CFTR locus following delivery of Cas12a RNPs using four different peptides. Shuttle peptides were tested for Cas12a RNP delivery using gRNA targeting CFTR intron 22–23. Materials were applied for 15 min, cells were harvested 72 hr later for Surveyor assay; Indel% determined by Sanger sequencing. Asterisks denote bands observed with gene editing. Np indicates Cas12a RNP with no peptide. c) S10 peptide dose–response on Cas12a RNP editing of CFTR locus. HAE transduced with fixed RNP concentration [Cas12a]: 1.33 µM; [gRNA]: 2 µM and S10 peptide concentrations varied (20–50 μM). Cells incubated with peptide-RNP for 15 min, and harvested 72 h later for Surveyor assay (Control: Cas12a RNP alone). D) Effect of incubation time and repeated of peptide-Cas12a RNP delivery on editing. [S10]: 40 μM; [RNP]: 40 µM, applied for indicated times. After 72 h, cells prepped for Surveyor assay and Sanger sequencing (Np indicates Cas12a RNP with no peptide, incubated for 3 h; Rpt denotes repeated application of peptide/RNP × 3 daily doses). n = 3 donors. E) Schematic of HPRT1 locus and Cas12a guide RNA target sequence along with editing efficiency on delivery of RNPs. Screen of four peptide formulations at 40 μM concentration, [RNP]: 2.5 µM; [gRNA]: 2.0 μM on primary HAE. Indicated peptide-RNP applied for 3 h; 72 hr later, cells were processed for Surveyor assay. Asterisks denotes genome editing. n = 3 donors. F) Schematic of the CFTR locus and Cas9 guide target (exon 11) and editing efficiency in HAE after Cas9 RNP delivery with each of four shuttle peptides. The same four peptide formulations were applied at [40 μM], with [RNP]: 2.5 μM; [gRNA]: 2.0 μM. Indicated shuttle peptide and Cas9 RNP applied for 3 h; 72 h later, cells processed for Surveyor assay. Asterisks denote genome editing. n = 3 donors. Pubmed