Codner, GF;
Mianne, J;
Caulder, A;
Loeffler, J;
Fell, R;
King, R;
Allan, AJ;
... Teboul, L; + view all
(2018)
Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.
BMC Biology
, 16
, Article 70. 10.1186/s12915-018-0530-7.
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Abstract
Background Recent 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. Results We 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. Conclusion lssDNA 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.
| Type: | Article |
|---|---|
| Title: | Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1186/s12915-018-0530-7 |
| Publisher version: | https://doi.org/10.1186/s12915-018-0530-7 |
| Language: | English |
| Additional information: | This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
| Keywords: | Science & Technology, Life Sciences & Biomedicine, Biology, Life Sciences & Biomedicine - Other Topics, Allele validation, Conditional, CRISPR/Cas9, Homologous recombination, Mouse, Mutant, Long single-stranded DNA, ONE-STEP GENERATION, HOMOLOGY-DIRECTED REPAIR, C57BL/6N MICE, GENE-FUNCTION, CRISPR, MANIPULATION, EFFICIENCY, PHENOTYPE, INSERTION, ALLELES |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10052827 |
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