CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells

Computational and Structural Biotechnology Journal
Gue-Ho HwangSangsu Bae

Abstract

CRISPR-Cas9 induces DNA cleavages at desired target sites in a guide RNA-dependent manner; DNA editing occurs through the resulting activity of DNA repair processes including non-homologous end joining (NHEJ), which is dominant in mammalian cells. NHEJ repair frequently causes small insertions and deletions (indels) near DNA cleavage sites but only rarely causes nucleotide substitutions. High-throughput sequencing is the primary means of assessing indel and substitution frequencies in bulk populations of cells in the gene editing field. However, it is difficult to detect bona fide substitutions, which are embedded among experimentally-induced substitution errors, in high-throughput sequencing data. Here, we developed a novel analysis method, named CRISPR-Sub, to statistically detect Cas9-mediated substitutions in high-throughput sequencing data by comparing Mock- and CRISPR-treated samples. We first pinpointed 'hotspot positions' in target sequences at which substitution mutations were quantitatively observed much more often (p > 0.001) in CRISPR- versus Mock-treated samples. We refer to the substitution mutations in defined hotspot positions as 'apparent substitutions' and ultimately calculated 'apparent substitution frequenci...Continue Reading

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Methods Mentioned

BETA
targeted
gene knock-ins
PCR

Software Mentioned

Amplican
CRISPResso2
Fastq
OFFinder
CRISPR
Django2
Sub
Python
SciPy
Cas

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