CRISPR Off-Target Editing Analysis

Demo

Amplicon deep-sequencing readout for a Cas9 guide: candidate off-target sites scored by mismatches and CFD, with edited-vs-control indel rates tested for significant editing above background (two-proportion test, Wilson 95% CIs, Benjamini–Hochberg FDR).

Analysis demo by Kalif Shear
CGT QC / Analytical Development

Guide RNA

FDR threshold (q)

q < 0.05

Min effect (Δ indel %)

Δ ≥ 0.5%

—

On-Target Editing

—

Guide Specificity Score

—

Candidate Off-Targets

—

Confirmed Off-Targets

Off-Target Editing vs. Matched Control

Indel frequency at each candidate site (whiskers = Wilson 95% CI). Sites where edited editing is significantly above the matched control are flagged as confirmed off-targets.

Edited sample Control sample Confirmed off-target

Site-Level Results

On-target shown as the positive control (top). Off-target candidates ranked by editing effect. Protospacer aligned to the on-target 20-mer; mismatches in red, PAM in grey.

Site	Protospacer (5'→3')	MM	CFD	Edited indel % (95% CI)	Control %	Δ%	p	q (BH)	Call