title: Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.
abstract: BACKGROUND
  The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations.
  
  
  RESULTS
  We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward.
  
  
  CONCLUSIONS
  The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Isabel
authors.initials: I
authors.lastname: Brocal
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: White
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dooley
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Samantha N
authors.initials: SN
authors.lastname: Carruthers
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Clark
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Amanda
authors.initials: A
authors.lastname: Hall
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
authors.forename: Elisabeth M
authors.initials: EM
authors.lastname: Busch-Nentwich
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. ds4@sanger.ac.uk.
authors.forename: Derek L
authors.initials: DL
authors.lastname: Stemple
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. rkettleborough@twistbioscience.com.|Twist Bioscience, 455 Mission Bay Boulevard South, San Francisco, CA, 94158, United States. rkettleborough@twistbioscience.com.
authors.forename: Ross N W
authors.initials: RN
authors.lastname: Kettleborough
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D054643:INDEL Mutation; D008297:Male; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013094:Spermatozoa / Q000166:cytology*; D015027:Zebrafish / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; Cas9; Embryo; Genome editing; Germline; Mutagenesis; Next generation sequencing; Perl; Zebrafish