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. 2016 Jul 12;34(7):695-7.
doi: 10.1038/nbt.3583.

Analyzing CRISPR genome-editing experiments with CRISPResso

Luca Pinello  1   2 , Matthew C Canver  3   4   5 , Megan D Hoban  6 , Stuart H Orkin  3   4   5   7 , Donald B Kohn  6 , Daniel E Bauer  3   4   5 , Guo-Cheng Yuan  1   2
Affiliations

Analyzing CRISPR genome-editing experiments with CRISPResso

Luca Pinello et al. Nat Biotechnol. .
No abstract available

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Quantification and visualization of NHEJ and HDR mutagenesis profiles
a–d, An example of NHEJ-mediated disruption of a coding sequence by CRISPR-Cas9 (experiment 1). a, Quantification of editing frequency as determined by the percentage and number of sequence reads showing unmodified and modified alleles. When no donor sequence is provided, CRISPResso classifies any mutation overlapping a window around the expected cleavage site/s as an NHEJ event. b, Frequency distribution of alleles with indels (shown in blue) and without indels (in red). Length-conserving substitutions are not classified as indels in this plot. In this example, the indels are dominated by small deletions, consistent with the anticipated CRISPR-Cas9 effect. c, NHEJ reads with insertions (red), deletions (purple), and substitutions (green) mapped to reference amplicon. For insertions, the positions immediately adjacent to the insertion are indicated. In this example, the mutations cluster around the predicted cleavage position, consistent with the anticipated CRISPR-Cas9 effect. A low level of substitutions apparent throughout the amplicon suggests low-level technical error, although these errors do not contribute to the quantification of the NHEJ. d, Frameshift analysis of coding sequence reads affected by modifications. Frameshift and in-frame mutations include any mutations that partially or fully overlap coding sequences as input by the user, with any non-overlapping mutations classified as noncoding (see also Supplementary Fig. 11). e–f, An example of HDR-mediated recombination of an extrachromosomal donor sequence resulting in four substitutions relative to the reference amplicon (experiment 2). e, When an expected HDR amplicon is provided, CRISPResso classifies sequence reads as HDR if they preferentially align to the expected HDR amplicon sequence and NHEJ (or unmodified) if they preferentially align to the reference amplicon. An alignment threshold may be provided to distinguish HDR alleles from those showing evidence of mixed HDR-NHEJ repair. f, Mapping of mutation position to reference amplicon of reads classified as NHEJ (left), HDR (center), and mixed HDR-NHEJ (right). In this example with the alignment threshold set to 100% sequence identity, the HDR alleles show only the four expected substitutions (see Supplementary Fig. 4) while the mixed HDR-NHEJ alleles show additional indels at the predicted cleavage position, consistent with sequential cleavages initially repaired by HDR and subsequently by NHEJ.
See this image and copyright information in PMC

References

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