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.

The cardiac response to myocardial injury includes fibrotic and hypertrophic processes and a key mediator in this response is transforming growth factor-β1 (TGF-β1). Caveolin-1 (cav1), the main structural protein of caveolae, is an inhibitor of the TGF-β1 signaling pathway. To examine the role of cav1 in cardiac repair, cav1 deficient (Cav1−/−) and wild type (WT) mice were subjected to cryoinjury of the left ventricle (LV). At baseline the two groups exhibited no inflammation, similar collagen content, and similar cardiac function. After injury, Cav1−/− animals displayed enhanced TGF-β1 signaling, as reflected by a 3-fold increase in the activation of the Smad2-dependent pathway and more widespread collagen deposition in the heart. Qualitative and quantitative analysis indicated that collagen deposition peaked in the WT LV 14 days after injury, accompanied by increased mRNA abundance for procol1a2 (2-fold) and procol3a1 (3-fold). Collagen deposition was further enhanced in Cav1−/− mice, which was accompanied by reduced expression of matrix metalloproteinases MMP −8 (3-fold) and −13 mRNA (2-fold). The levels of expression of inflammatory markers of acute phase were similar between the strains However, macrophage clearance in the damaged region was delayed in Cav1−/− mice. We observed a 4-fold decrease in collagen deposition in Cav1−/− mice injected with a cav1 scaffolding domain peptide (CSD) and a 2-fold decrease in WT mice treated with the CSD. We conclude that cav1 has a direct role in reducing TGF-β1 signaling and as such might be an appropriate target for therapies to influence cardiac remodeling.

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Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.