The bacterial CRISPR-Cas9 system has been repurposed for genome engineering in eukaryotic cells. How does the CRISPR-Cas9 system find specific target in the genome not well defined in living cells. It has been shown that the CRISPR-Cas9 genome interrogation is subjected to 3D diffusion in living cells (Knight et al., Science 2015), and the searching process is slow but in a Cas9/guide RNA dose-dependent manner (Jones et al., Science 2017). We have found that CRISPR guide RNA is extremely unstable in the absence of Cas9. Meanwhile CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time (Ma et al., JCB 2016).
Prime editing developed by David Liu’s lab is a very promising tool for base substitution, small fragment insertion and deletion with high precision (Anzalone et al., Nature 2019). We have found the pegRNAs are extremely unstable even in the present of Cas9 during prime editing. We are applying for the above principles to stabilize pegRNA, optimize Cas9/pegRNA assembly, improve DNA interrogation and prime editing.