[HTML][HTML] Off-target mutations are rare in Cas9-modified mice

V Iyer, B Shen, W Zhang, A Hodgkins, T Keane… - Nature …, 2015 - nature.com
V Iyer, B Shen, W Zhang, A Hodgkins, T Keane, X Huang, WC Skarnes
Nature methods, 2015nature.com
To the Editor: The clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas
system is a highly efficient technology for genome editing of mouse zygotes1. Previously, we
reported cotransmission of a Cas9-induced mutation in the X-linked Ar gene and an off-
target mutation to offspring of founder animals from pronuclear injection of Cas9 mRNA and
two single guide RNAs (sgRNAs) 2. No off-target damage is observed in offspring derived
from founder animals injected with Cas9 nickase mRNA and a pair of sgRNAs, showing that …
To the Editor: The clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas system is a highly efficient technology for genome editing of mouse zygotes1. Previously, we reported cotransmission of a Cas9-induced mutation in the X-linked Ar gene and an off-target mutation to offspring of founder animals from pronuclear injection of Cas9 mRNA and two single guide RNAs (sgRNAs) 2. No off-target damage is observed in offspring derived from founder animals injected with Cas9 nickase mRNA and a pair of sgRNAs, showing that CRISPR-Cas specificity is improved by employing nickases2, 3. Here, we used whole-genome sequencing to more thoroughly assess any damage induced by injection of Cas9 versus Cas9 nickase.
Two female founder animals (C57BL/6J× CBA) carrying biallelic deletions in Ar induced with Cas9 and Cas9 D10A nickase (animals F18 and F25, respectively) were mated to C57BL/6J males, and heterozygous Ar mutant offspring were sequenced. To control for strain-specific variants, we also sequenced a C57BL/6J and a CBA animal from our breeding colonies. Whole-genome sequencing was performed at a sufficient depth (20–25×) to detect more than 95% of
nature.com