CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.
CRISPR Gene Editing