Validation of CRISPR/Cas Experiments

Package Overview

Goal

Validation of CRISPR/Cas9 experiments at one or more regions of interest

Requirements

  • Each region of interest must be PCR-amplified with specific primers
  • PCR amplicons from multiple regions can be pooled, provided they are non-overlapping
  • PCR amplicons from multiple samples must not be pooled
  • Sequencing must cover the entire amplicon
  • When doing paired-end sequencing, reads must overlap by at least 20bp
  • Maximum amplicon length depends on sequencing strategy:
    • 100bp paired-end: 180bp
    • 150bp paired-end: 280bp
    • 250bp paired-end: 480bp
    • 300bp paired-end: 580bp
  • Minimum coverage: 50 reads per amplicon

Analysis

  • Application of Crispresso2 (Clement et al. 2019, see download):
    • Mapping of reads to the amplicons
    • Quantification of substitutions and/or indels
    • Classification of indels in open-reading frames into in-frame and frame-shift
    • In case of homology-directed repair, quantification of the alternative allele at the CRISPR/Cas9 cut site

Output

  • HTML report per sample with quantification and visualization of the editing events
  • Summary HTML report with an overview of all samples

Advanced Analysis

Not available

References

Clement, Kendell, Holly Rees, Matthew C Canver, Jason M Gehrke, Rick Farouni, Jonathan Y Hsu, Mitchel A Cole, et al. 2019. “CRISPResso2 Provides Accurate and Rapid Genome Editing Sequence Analysis.” Nature Biotechnology 37 (3): 224–26. https://www.nature.com/articles/s41587-019-0032-3.