CRISPR-CLEAR: Nucleotide-Resolution Mapping of Regulatory Elements via Allelic Readout of Tiled Base Editing

Abstract

CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR, 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. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.

Figure 1:. A base-editor tiling screen with allele-based readout

a. Comparison of CRISPR-CLEAR workflow with standard sgRNA enrichment sequencing approach. The workflow illustrates the key steps from guide RNA design to data analysis. First, cells stably expressing a base editor are transduced with a library of guide RNAs tiling the regulatory sequence. After editing, cells are FACS-sorted based on the expression of the target protein. Genomic DNA is extracted from sorted cells. Next-generation libraries are prepared to quantify sgRNA counts and to measure the distribution of edits at the endogenous sequence in the sorted population of cells. The left pathway shows the standard approach using sgRNA count-based readout and the CRISPR-SURF pipeline for deconvolution of functional regions. The right pathway depicts the CRISPR-CLEAR approach using direct allele-based readout and the CRISPR-Millipede pipeline, enabling precise genotype-to-phenotype linkage through per-allele and per-nucleotide analysis. b. A putative proximal enhancer is located upstream of the CD19 promoter, based on H3K27ac and ATAC-seq of NALM6 cells, and sequence conservation. c. NALM6 clones with mono- or biallelic deletion of the CD19 enhancer show reduction in CD19 mRNA levels. d. Wild-type and enhancer KO clones were stained with a CD19 antibody. Flow cytometry indicated a reduction in CD19 protein levels. One-way ANOVA, replicates are shown in circles/squares/triangles (n=6–10), * = p<0.05).

Figure 2:. Direct measurement of regulatory potential using allele-based readout

a. CRISPR-SURF analyses. Top: plot showing the fold enrichment of sgRNA in CD19 positive versus CD19 negative cells in the ABE8e-SpRY screen. Scores of guides from each of three replicates are shown in red, blue, or green. Scores of negative control sgRNAs (20mers not containing editable adenines) are shown in grey. Deconvolution score track with the region called significant at positions 220–230 by CRISPR-SURF demarcated with a black segment below. Bottom: plot showing the fold enrichment of guides from the evoCDA screen. Negative control guides (20mers lacking a cytosine within the expected editing window, with a non-NGG PAM) are shown in grey. Deconvolution score track with the regions called as significant at positions 140–150 and 220–230 by CRISPR-SURF demarcated with a black segment below. b. CRISPR-Millipede analyses. From top to bottom: plot showing the effect sizes obtained for A>G (green) and T>C (purple) substitutions (covariates) at given positions in the sequence from the CRISPR-Millipede analysis of the alleles in the CD19 positive and CD19 negative sorted populations from the ABE8e-SpRY screen. Positive effect size indicates variants leading to lower CD19 expression. Track showing the posterior inclusion probabilities (PIP) for each of the covariates in the ABE8e-SpRY screen. Track showing the editing rate of A>G and T>C substitutions in the CD19 positive (red) and CD19 negative sorted populations (blue). Plot showing the effect sizes obtained for G>A (yellow) and C>T (blue) substitutions (covariates) at given positions in the sequence from the CRISPR-Millipede analysis of the alleles in the CD19 positive and CD19 negative sorted populations from the evoCDA screen. Track showing the posterior inclusion probabilities (PIP) for each of the covariate in the evoCDA screen. Track showing the editing rate of G>A and C>T substitutions in the CD19 positive (red) and CD19 negative populations (blue). Two regions with overlap of hits in CRISPR-SURF and CRISPR-Millipede are highlighted as pink (region 1) and light blue (region 2). c. Top: TF motifs found at sequences with regulatory potential (high betas). Motifs were filtered based on the expression of the cognate transcription factors in NALM6 cells (>25 CPM) and significant genome-wide CRISPR screen scores (>0.5 or <−0.5) for modulation of CD19 from a past study. Bottom: PhyloP scores for the CD19 enhancer show that CRISPR-Millipede hits are highly conserved. d. PyDESEQ2 analysis: Differentially enriched alleles in CD19 negative or high populations in both screens. Pink dots correspond to alleles containing identified CRISPR-Millipede hits in region 1 (151A>G in the ABE8e-SpRy screen and 154G>A in the evoCDA screen). Light blue dots correspond to alleles containing identified CRISPR-Millipede hits in region 2 (223A>G, 230A>G in the ABE8e-SpRy screen, 227T>C, 229T>C in the evoCDA screen). Representative alleles are labeled. e. CRISPR-Millipede visualizations from top to bottom: board plot highlighting estimated nucleotide level effects on region 2 (chr16:28930891–28930931). The visualization consists of a heatmap showing CRISPR-Millipede effect sizes (square color), PIP (square size) and WT nucleotide with circles for the ABE8e-SpRy screen. Top substitutions with high effect size and PIP include 223A>G and 230A>G. Board plot for the evoCDA screen. Top substitutions with high effect size and PIP include 227T>C and 229T>C. Track showing the reference sequence for region 2. Track showing recovered effect sizes for both screens as logo track. Tracks showing candidate TF motifs including SPIB, IKZF1, and PAX5. Tracks showing the editing rate of A>G and T>C and the C>T and G>A substitutions in the two screens (Red: CD19 positive, Blue: CD19 negative).

Figure 3:. Targeting Millipede hits downregulates CD19 and provides resistance to aCD19-CAR T cells.

a. Map of the recovered hits by CRISPR-Millipede on the CD19 enhancer sequence, showing TF binding motifs and sgRNAs used for validation experiments. Editable nucleotides are shown in green. b. Validation using individual sgRNAs and flow cytometry. While sg119, targeting a neutral sequence within the enhancer showed no effect, editing with sgRNAs 145, 217, 218, 220, 223 and 225 results in downregulation of CD19 MFI compared to sgAAVS1. c. Millipede analysis using sg218 highlights nucleotides 220, 223, 224 and 230. d. Genomics tracks of the CD19 locus: the CD19 enhancer is occupied by IKZF1 and PAX5. e. Targeting PAX5 and SPIB, but not MYB, resulted in downregulation of CD19. f. Schematics of the CAR-T co-culture experiments. Wild-type, BFP+ NALM6 cells are mixed 1:1 with GFP+ NALM6 cells carrying edits at the CD19 enhancer and co-cultured with aCD19 CAR T cells or mock T cells. High GFP/BFP ratio indicates that editing facilitates resistance to aCD19-mediated killing. g. NALM6 cells edited with sg145 are resistant to aCD19 CAR T. h. sg218 provides a milder, yet significant, accumulation of aCD19-resistant cells. One-way ANOVA, replicates are shown in circles (n=3), ** = p<0.01; *** = p<0.001; **** = p<0.0001 (b, e). Multiple unpaired t test, replicates are shown in circles (n=8–10), **** = p<0.000001 (g, h).