Fig. 2: GUIDE-tag enables genome-wide off-target analysis in vivo.
From: Genome-wide detection of CRISPR editing in vivo using GUIDE-tag
a Schematic of GUIDE-tag editing outcomes and strategies to create UDiTaS or GUIDE-tag Illumina sequencing libraries, where insertion is the DNA tag. Tn5 enzyme tagments genomic DNA and adds UMI, pooling barcode, and i5 primer sites. Desired genomic regions are amplified with i5 Primer and a viewpoint specific primer: for UDiTaS a locus-specific genomic forward or reverse (Locus_F or R) and for GUIDE-tag a tag-specific forward or reverse (Insert_F and R). A second round of PCR adds the i7 adaptor sequence. b Quantification forward/reverse donor insertion and indels (Locus_F) at the Fah target site in biotin-dsDNA plus Cas9-mSA+sgFah treated animals by UDiTaS. Genomic DNA was collected from NTBC on D0 and D34 mice in Fig. 1. Values are mean ± SD (n = 3 mice each group). * P < 0.05 by unpaired, two-tailed Student’s t-test. c In vivo off-target (OT) sites identified by GUIDE-tag in biotin-dsDNA plus Cas9-mSA+sgFah treated animals. Mismatches relative to the target site (On) are shown with colored boxes. UMI numbers for each site are shown (average of three mice). 1,2,3 are three different mice with indel rates indicated as a heatmap. d Venn diagram of sgFah off-target sites identified by GUIDE-tag or by CRISPRseek prediction. e Scatter plot shows correlation between unique UMI% and indel rates at the 7 sgFah off-target sites (average of 3 mice). Dashed lines represent the linear regression fit (Pearson correlation calculated). The p-value for Pearson’s correlation coefficient was determined by the two-tailed t-distribution table. Dots on Y-axis represent UMIs for each OT for each of 3 mice. Source data are provided as a Source Data file.
