Deep mutational scanning of a CRISPR-Cas13d protein

The goal is to characterize the functional effects of single amino acid mutations across the catalytically deactivated CRISPR-Cas13d protein (dCasRx) on splice modulation activity. A deep mutational scanning (DMS) approach was used to systematically assess variant function. Functional splicing activity was measured using a massively parallel reporter assay in a K562 cell line stably expressing a bichromatic fluorescent reporter of alternative splicing. Variant abundance was quantified by amplicon sequencing of targeted sections of the dCasRx coding region from sorted cell populations. The deep mutational scan was performed with 8 sublibraries, where each sublibrary targeted a different region of the dCasRx coding sequence. Cells were sorted into 4 bins based on DsRed fluorescence, where higher fluorescence indicated higher dCasRx-mediated splicing activity. Bin numbers were assigned such that bin 1 corresponds to the bottom 25% fluorescent bin, whereas bin 4 corresponds to the top 25% fluorescent bin. Functional activity scores were derived based on the enrichment across the four sorted bins.