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Graham McVicker

@grahammcvicker.bsky.social

Associate Professor at the Salk Institute

Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

Superb-seq is also EASY TO PERFORM. Protocol uses standard kits, equipment, and NO virus. We also provide free software, SHERIFF, to analyze Superb-seq data. We hope this will enable wide-spread adoption and use in diverse cell types!

On-target edit events detected for different cell types (K562, primary T cells, GM12878, Jurkats) and guides using TIDE. Most tested guides resulted in efficient insertions of the 34bp donor sequence containing the T7 promoter.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

All off-targets were non-coding, so paired scRNA-seq was crucial to determine their functional effects using differential expression, as shown for the USP9X intronic edit above.

Edit events colored by the corresponding guide. Only one guide had no detected off target events. Y-axis shows homology between genome and guide spacer. X/I/O indicates whether edit is exonic, intronic, or intergenic. Most off-target edits are intronic.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

Many of the off-target edit sites (total = 36) were not predicted by popular in silico tools. These tools appear to underestimate the tolerance of guide bulges and mismatches with off-target edit sites, yet also predict many off-target edits that do not occur.

Venn diagram showing overlap between off-target edit sites identified with Superb-seq and those predicted by in silico tools Cas-OFFinder, COSMID and E-CRISP. The overlap is poor and 11 off-targets were not predicted by any computational tool.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

Off-target sites had clear guide homology and PAMs. There was however a surprising tolerance of guide bulges and mismatches with off-targets, including mismatches in the “seed” region.

Pairwise alignments of SMARCA4 g22 sequence and the genome site at the edit. All off-target sites have PAMS, but have various mismatches to the guide spacer sequence.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

One guide had an off-target edit that was 34x more frequent than the on-target edit! A total of 5 off-target sites were observed in more cells than the on-target for this particular guide.

Detected edits for SMARCA4 guide 22. 13 edits were detected. Y axis is the frequency of the edit (number of cells) and X axis is the homology between the genome and the guide spacer sequence. Some off target events are more frequent than the intended on-target.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

We quantified Cas9 edits per cell (‘edited alleles’), and associated edit alleles with gene expression. One intronic off-target edit perturbed the expression of USP9X and >100 downstream genes!

Sheriff counts edit alleles per cell at each edit site. An off-target edit within the first intron of USP9X is associated with differential expression of USP9X and >100 downstream genes.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

SURPRISING RESULT. We applied Superb-seq to 10k K562 cells and detected pervasive off-target Cas9 edits, with an average of 6 off-target sites per guide, ranging in frequency from 0.03-18.6% of cells!

Genome positions and frequencies (number of cells) of on and off-target events detected with Superb-seq.

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Graham McVicker @grahammcvicker.bsky.social · 11/02/2025

Superb-seq detects edits and cell RNA by labelling nuclease cleavage sites with homology-free insertion of a T7 promoter, followed by “Zombie” in situ transcription with T7-pol to generate edit-site marking barcoded RNA. T7 and cell RNAs are jointly read out with scRNA-seq.

A schematic of Superb-seq showing 3 steps: (1) edit labeling with donor sequence containing T7 promoter; (2) In situ transcription with T7 polymerase; (3) combinatorial single-cell RNA-seq + computational analysis with Sheriff.

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