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!

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.

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.

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.

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.

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!

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!

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.