Journal: Nucleic Acids Research

Article Title: Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

doi: 10.1093/nar/gkac095

Figure Lengend Snippet: Peptide nucleic acids efficiently hybridize gRNA spacer sequences in vitro . ( A ) Structures of DNA and PNA oligomers. ( B ) Schematic depicting antisense PNA hybridization to a Cas9 sgRNA spacer sequence. ( C ) Melting curves for DNA:RNA (blue), RNA:RNA (gray) and PNA:RNA (red) 20-bp duplexes. Data are shown as normalized OD (260nm) measurements across temperatures. ( D ) Electrophoretic mobility shift assay (EMSA) assay demonstrating antispacer PNA binding to sequence-matched sgRNA and Cas9 RNP on polyacrylamide gel . Cas9 RNP band is labeled. ( E ) Antispacer PNA binding to Cas9 RNP loaded with BFP sgRNA (blue) and HBB sgRNA (red), described as TAMRA band integrated density from analysis of gels from three independent experiments. For (E), bars represent mean ± s.d. from n = 3 independent experiments, mean differences and P -values listed in .

Article Snippet: TAMRA dye-labeled PNA oligomers were synthesized manually on 10% l -lysine loaded solid support (MBHA (4-methylbenzhydrylamine) resin, Peptides International, RMB-1045-PI) using standard Boc chemistry procedures.

Techniques: In Vitro, Hybridization, Sequencing, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling