Nature communicationsMar 2, 2026

Mapping gene control regions at single DNA letter resolution using targeted DNA editing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

Tiled base editing mapped CD19 enhancer nucleotides whose mutation can lower CD19 and enable CAR-T escape.

Evidence

Proof-of-concept regulatory-element mapping combined dense base-editing mutagenesis, targeted allele sequencing, motif analysis, and validation at a putative CD19 enhancer.

Caveat

The abstract tests one enhancer and immunotherapy context, so generality to other regulatory elements or clinical resistance remains unshown.

Simplified

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.