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Fig. 1 | Genome Biology

Fig. 1

From: iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity

Fig. 1

Diagram of the method, illustrating the strategy for improving specificity and examples of output. Procedure for GUIDE-seq (a) and iGUIDE (b). A dsODN is incorporated into DNA breaks. Amplification of flanking DNA, by nested-PCR, produces sequence copies indistinguishable from genomic mispriming when using the GUIDE-seq design. The modified dsODN of iGUIDE uses a reporter present in sequence output to identify correctly primed molecules. c Alignment of amplification primer and upstream sequence from uniquely identified sites in either GUIDE-seq or iGUIDE samples. We reasoned that amplification products resulting from mispriming should be just adjacent to sequences in the human genome with adventitious matches to the amplification primer sequence. Evidence for greater matching to primer sequences in a sample thus provides evidence for more mispriming. In the figure, the x-axis scores the match of the inferred human flanking DNA to the amplification primer (marked 2 in a and b); higher numbers of matching bases than seen for random sequences (light red) indicates probable mispriming. P values compare the distributions of the matches to the primer sequences in DNA samples detected for GUIDE-seq (top) and iGUIDE (bottom). d Sequence coverage of an on-target CRISPR site from iGUIDE data (gRNA targeting B2M)

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