Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Abstract

BackgroundRecent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.ResultsWe generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors.ConclusionlssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0530-7) contains supplementary material, which is available to authorized users.

Clarin‐2 is essential for hearing by maintaining stereocilia integrity and function

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Simple embryo injection of long single‐stranded donor templates with the CRISPR/Cas9 system leads to homology‐directed repair in Xenopus tropicalis and Xenopus laevis

“…Here we injected lssDNA, not dsDNA, and we do not know how lssDNA behaves in injected Xenopus embryos as opposed to injected dsDNA that can persist (at least somatically) in adults and often exists as tandem head‐to‐tail concatemers integrated in the genome (Etkin & Pearman, 1987 and references therein). In mice, it has been shown that random insertions of injected lssDNA into the genome could also occur (Codner et al, 2018). Because of these results, we tested whether any evidence of germline transmission of randomly integrated lssDNA can be observed in albino embryos obtained from the F0 “Female 1” crossed with an albino male.…”

Simple Protocol for Generating and Genotyping Genome‐Edited Mice With CRISPR‐Cas9 Reagents

“…The first thing to take into account after an experiment involving CRISPR-Cas9 tools is the genetic noise (variability) produced by the endogenous repairing systems, particularly the non-homologous end joining pathway. No matter what you do, or what tricks you implement, you are going to be dealing with mosaic founder mice, whose complexity can vary enormously (Codner et al, 2018;Seruggia, Fernández, Cantero, Pelczar, & Montoliu, 2015). The CRISPR-Cas9 reagents, deposited (microinjected or electroporated) in mouse fertilized oocytes, will remain active for several cell divisions.…”