SpRY-mediated CRISPR screening facilitates functional dissection of non-coding sequences at single-base resolution

Interpretation of the non-coding genome and genetic variants has been facilitated by epigenetic mapping of cis-regulatory elements and functional characterization in their native chromatin contexts. CRISPR perturbation screens with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants, but at limited resolution due to absence of protospacer adjacent motif (PAM) sequences. Here, we developed CRISPR mutagenesis screens using the near-PAMless SpRY variant, which enables interrogation of the non-coding genome at single-base resolution. Using this approach, we identified the proximal regulatory elements necessary for target gene expression and known pathogenic variants in the genes encoding S19 (RPS19) and the ferritin light chain (FTL) as proof-of-concept examples. The approach described here is generally applicable to functional interrogation of all non-coding genomic elements, and complements other high coverage CRISPR screens.