CRISPR-Cas9 Activities with Truncated 16-Nucleotide RNA Guides Are Tuned by Target Duplex Stability Beyond the RNA/DNA Hybrid

Abstract

CRISPR-Cas9 has been adapted as a readily programmable genome manipulation agent, and continuing technological advances rely on an in-depth mechanistic understanding of Cas9 target discrimination. Cas9 interrogates a target by unwinding the DNA duplex to form an R-loop, where the RNA guide hybridizes with one of the DNA strands. It has been shown that RNA guides shorter than the normal length of 20-nucleotide (-nt) support Cas9 cleavage activity by enabling partial unwinding beyond the RNA/DNA hybrid. To investigate whether DNA segment beyond the RNA/DNA hybrid can impact Cas9 target discrimination with truncated guides, Cas9 double-stranded DNA cleavage rates (k cat ) were measured with 16-nt guides on targets with varying sequences at +17 to +20 positions distal to the protospaceradjacent-motif (PAM). The data reveal a log−linear inverse correlation between k cat and the PAM+(17−20) DNA duplex dissociation free energy (ΔG NN(17−20) 0 ), with sequences having smaller ΔG NN(17−20) 0