A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

RNAi knockdown of the flightless-I transcript in Drosophila melanogaster

Thesis (M.S.)--University of Hawaii at Manoa, 2007.Includes bibliographical references (leaves 63-69).69 leaves, bound ill. (some col.) 29 c

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination

International audienc

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

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

Additional file 18: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S16. Generation of a point mutation in Rims1 with ssODN donors. (a) The table details the F0 animals obtained for generation of Rims1 mutant with ssODN donors. The ID and outcome of sequencing the region of interest, as well as the conclusion for each individual are shown. (b) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from the F0 animals. Sequences of Rims1-ODN-151 mosaic and of sub-cloned amplicons are shown in Additional file 3: Figure S2u and v, demonstrating the presence of the desired mutation in this animal that was therefore mated. (c) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from Rims1-ODN-151’s offspring. Animal IDs are shown. + is positive control amplified from an unrelated WT animal. L1 = 1 kb DNA molecular weight (thick bands are 3 kb); L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). (d) The table details the first litter obtained by mating Rims1-ODN-151 with a WT mouse. The ID, outcome of sequencing the region of interest and copy counting of the region of interest as well as the conclusion for each individual are shown. Sequencing of Rims1-ODN-151.1g is shown in Additional file 3: Figure S2w and illustrates the failure of transmission of the desired allele. (PNG 893 kb

Additional file 20: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

The file contains the raw sequencing data obtained from the founders generated for the Rims1R655H point mutation. (ZIP 21747 kb

Additional file 17: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S15. Design of a Rims1R655H point mutation. The figure illustrates the changes designed at the nucleotide and proteomic levels with the mutagenesis strategy employing (a) oligonucleotides and (b) lssDNA. Coding sequences are translated into protein sequences above annotated exon. Note that the region containing Rims1 is not entirely accurate in the GRCm38 assembly. We have re-sequenced this region prior to designing of the mutant (primers shown in Additional file 1: Table S1). (PNG 521 kb

Additional file 10: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S9. Analysis of the 6430573F11Rik project. PCR amplification of genomic DNA of (a) F0 animals, (f) 6430573F11Rik-11’s offspring or (i) 6430573F11Rik-28’s offspring with (a, f) 6430573F11Rik-F3 and 6430573F11Rik-R2 (1721-bp amplicon) and (b, f) LoxPF and LoxPR (999-bp amplicon). Sequencing of PCR amplicons from (c) 6430573F11Rik-11 and (g) 6430573F11Rik-11.1a with 6430573F11Rik-F3 and 6430573F11Rik-R2. LoxPs are in blue. ID, outcome of PCR analysis and conclusion for (d) each F0 animal and (e) the first litter obtained by mating 6430573F11Rik-11 with a WT mouse. Two founders were mated for cKO GLT. *Mated; ⁑no evidence of loxP in 6430573F11Rik amplicon, suggesting donor integrated randomly (6430573F11Rik-28 sequence trace in Additional file 3: Figure S2q). (g) Only WT sequence is found, indicating random donor insertion. (f, i) Animal IDs are shown. + is positive control from unrelated WT and conditional floxed animal for 6430573F11Rik and LoxP PCR, respectively. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (h) First litter obtained by mating 6430573F11Rik-28 with a WT mouse. ID, outcome of sequencing and copy counting of the region of interest and the conclusion for each individual. (j) Sequencing of amplicons obtained with 6430573F11Rik-F3 and 6430573F11Rik-R2 and 6430573F11Rik-28.1a. Only WT sequence is found, indicating random donor insertion. Sequencing of deletion allele in founder 6430573F11Rik-6, summary of analysis of F1 animals derived from 6430573F11Rik-6 and transmitted deletion allele are shown in Additional file 3: Figure S2r, s and t. (PNG 1011 kb

Additional file 2: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S1. Screening by Sanger sequencing of animals for the generation of a Syt7 conditional allele. The figure shows the sequencing traces from PCR products amplified from founder Syt7-4 (a) and founder Syt7-8 (b) that reveal the integration of two loxP sites in both animals. Note that Syt7-8 appears to be homozygous (a single trace detected), while Syt7-4 appears to contain at least two different alleles. The PCR products from which the sequence traces were derived are shown in Fig. 1. (PNG 377 kb

Additional file 13: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S12. Examples of unexpected point mutations in the F0 animals obtained from the co-injection of CRISPR/Cas9 reagents and lssDNA in 6430573F11Rik (a) and Cx3cl1 (b and c) projects. Blue 5′ homology arm; orange universal sequences for diagnostics; green critical region with exon in capitals; red loxP sites; grey 3′ homology arm. Unexpected point mutations are detected by Sanger sequencing of amplicons generated with primers external to the donor; (a) shows one intronic SNP in floxed critical region, (b) shows two intronic nucleotide changes (black arrows, grey highlight) and one coding nucleotide change (red arrow, pink highlight) which was found associated with (c) SNP in 3’ loxP site. Mutations are highlighted on the sequence alignment (a) and seen on the sequence chromatograms (b and c). (PNG 1332 kb