Fig. 2: Experimental screening of the activity of KKH-SaCas9 variants. | Nature Communications

Fig. 2: Experimental screening of the activity of KKH-SaCas9 variants.

From: Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities

Fig. 2: Experimental screening of the activity of KKH-SaCas9 variants.

a, b Strategy for the profiling of the activities of KKH-SaCas9 variants in human cells is illustrated in (a). A library of 1,296 KKH-SaCas9 variants was assembled by PCR-based mutagenesis and was cloned in tandem with a gRNA-targeting GFP expressed from a U6 promoter. The library was delivered via lentiviruses to OVCAR8-ADR reporter cell lines in which the RFP and GFP genes are expressed from UBC and CMV promoters, respectively. Fluorescent protein expressions were analyzed by flow cytometry (results are shown in (b)). The activity of KKH-SaCas9 was measured using reporter systems in which the gRNA spacer sequence completely matched the GFP target site. Cells with an active KKH-SaCas9 variant were expected to lose GFP fluorescence. Cells were sorted into bins, each encompassing ∼5% of the population based on GFP fluorescence, and their genomic DNA was extracted for quantification of the variant by Illumina NovaSeq. c–e Scatterplots comparing the barcode count of each KKH-SaCas9 variant between the bin-A (GFP-negative) and the bin- B (GFP-positive) populations. Each dot represents a KKH-SaCas9 variant, and wild-type (WT) KKH-SaCas9 is labeled. Solid reference lines denote 2-fold enrichment, and the dotted reference line corresponds to no change in barcode count in the bin-A as compared with the bin-B population. Three sgRNAs with permissive (sg1, sg2, and sg3) (c) and three sgRNAs with nonpermissive (sg5, sg6, and sg7) (e) PAMs for KKH-SaCas9 were used. Bubble plot summarizing the enrichment scores determined for each KKH-SaCas9 variant with the three sgRNAs with permissive PAMs is shown in (d).

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