Multi-locus CRISPRi targeting with a single truncated guide RNA

Abstract

A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.

Fig. 1. Truncated guides enable on-target CRISPRi-mediated repression.

a Schematic of truncated guide experiments. b CRISPRi in Jurkat cells with truncated CD81 promoter targeting guide and analyzed for CD81 cell surface expression. c Cas9 cleavage in Jurkat cells treated with two guides targeting CD81 and truncated versions. d CRISPRi in A375 cells with truncated CD81 promoter targeting guide and analyzed for CD81 cell surface expression. e Gene expression in A375 cells in 20nt and g[9nt] sgCD81i-1 CRISPRi populations. p-value cutoff at >2 and log fold-change >2 and <−2 using a linear mixed model and two-sided test that adjusts for multiple hypotheses. Significantly upregulated genes in 20nt and g[9nt] are 2 and 34, respectively. For b–d, cells were stained with CD81-FITC antibody analyzed by flow cytometry 7 days post lentiviral transduction for CD81 targeting guide, safe harbor control (Safe), or untransduced (UT). Flow gating strategy found in Supplementary Fig. 1f. Data are mean +/− SD from biological triplicate. Source data are provided as a Source Data file.

Fig. 2. Enhancer targeting with truncated guides induces EPB41 repression.

a Schematic of EPB41 genomic regulatory region and full-length/truncated guides targeting each of 4 TF motifs. b TF motifs (JASPAR) targeted by the guides with protospacer adjacent motif (PAM) orientation denoted. c Number of sequence match target sites in the genome (hg38) for the indicated motif directed guide. d Quantitative real time PCR analysis of EPB41 expression in K562 cells with respective guide CRISPRi treatment conditions. Safe (Safe Harbor-targeting) guide = g[20nt], promoter guide = 21nt, enh1 = g[20nt], enh2 = 21nt, enh3 = g[20nt] (enhancer guides from Gasperini et al. 2019). Data are mean +/− SEM. e Violin plot summary of EPB41 expression data in panel d organized by guide length or targeting region. TF-targeting guides (n = 16; 4 guides per spacer length), sgEnh (n = 12; 3 guides), and Promoter (n = 4; 1 guide, all points shown) are the product of 2 biological replicates per guide run in duplicate. Safe is the sum of 3 biological replicates run 2-3 times each (n = 8). Data are normalized to the mean CT value of Safe Harbor actin-b control probe. Results are analyzed by one-way ANOVA. ****P < 0.0001 for all guide lengths compared with Safe Harbor guide. P = 8.24 × 10⁻⁷, 9.95 × 10⁻⁷, 5.62 × 10⁻⁴, 3.98 × 10⁻⁵, and 0.01 for Safe vs. 21nt,g[20nt]; 14nt,g[13nt]; 11nt; sgEnh; and Promoter respectively. Source data are provided as a Source Data file.

Fig. 3. Simultaneous screening of CTCF binding sites with a truncated guide.

a Guide selection strategy based on top Homer generated motif from CTCF ChIP in Jurkat cells. b CTCF targeting schematic of a single 10nt guide directing CRISPRi to many motifs simultaneously (CTCF-bound and unbound). c Target site distribution (black) and CTCF occupancy based on Jurkat ChIP (blue) for each of the 10nt guide sequences. d, e Guide enrichment and depletion in pooled screen for the indicated cell lines. Pooled screens were run in triplicate, while K562 was in duplicate. Data are mean +/− SD. CTCF promoter targeting full length guide knockdown (KD) used as a control. Source data are provided as a Source Data file.

Fig. 4. CRISPRi with a single truncated guide disrupts CTCF binding at multiple loci.

a CTCF ChIP of the CTCF bound sites (134) targeted by sg4 in Jurkat cells. Significance with t-test ***P = 5.6 × 10⁻¹³. b H3K9me3 ChIP of sg4 targeted perfect match sites (357) with genomic bins plotted (416). Two bins were included in cases where an sg4 site targeted a bin boundary. Significance with t-test ***P = 1.3 × 10⁻³⁸. Data are aggregated from two independent replicates and presented as median values +/− 1 SD (box), with whiskers extending 1.5x below the first quartile and 1.5x above the third quartile in a and b. c Scatter plot CTCF binding at JASPAR motifs (880k) with 357 perfect match sites (black) and partial match sites (gray). d H3K9me3 density plot for Safe Harbor (S.H., light purple) and sg4 (dark purple) treated Jurkat cells. e Track view of a targeted region depicting CTCF and H3K9me3 signal from 2 replicate experiments (rep). Bars on the bottom depict CTCF perfect match (black) and partial match sites (gray). f dCas9 density plot for Safe Harbor (S.H., light purple) and sg4 (dark purple) treated Jurkat cells. g Volcano plot of CTCF bound sites (400k) with significant 10nt match CTCF sites (black), significant partial match sites (79), and not significant sites. Significance was determined at cutoffs (abs [LFC] > 0.5 and -log10 P > 5) using a linear mixed model and two-sided test that adjusts for multiple hypotheses. h Histogram depicting significant CTCF disrupted sites as a fraction of CTCF bound sites with 0 mismatches (black) up to 4 mismatches (blue). Raw values depicted on each bar. i Logogram of significant perfect match sites (top, 79 sites) and significant partial match sites (bottom, 79 sites) from Jurkat sg4 experiments. Source data are provided as a Source Data file.