title: Analysing the outcome of CRISPR-aided genome editing in embryos: Screening, genotyping and quality control.
abstract: The application of CRISPR/Cas9 technology has revolutionised genetics by greatly enhancing the efficacy of genome editing in the early embryo. Furthermore, the system has enabled the generation of allele types previously incompatible with in vivo mutagenesis. Despite its versatility and ease of implementation, CRISPR/Cas9 editing outcome is unpredictable and can generate mosaic founders. Therefore, careful genotyping and characterisation of new mutants is proving essential. The literature presents a wide range of protocols for molecular characterisation, each representing different levels of investment. We present strategies and protocols for designing, producing and screening CRISPR/Cas9 edited founders and genotyping their offspring according to desired allele type (indel, point mutation and deletion).
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Joffrey
authors.initials: J
authors.lastname: Mianné
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Gemma F
authors.initials: GF
authors.lastname: Codner
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Adam
authors.initials: A
authors.lastname: Caulder
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Fell
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Marie
authors.initials: M
authors.lastname: Hutchison
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Ruairidh
authors.initials: R
authors.lastname: King
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Stewart
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Wells
authors.affiliation: The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK. Electronic address: l.teboul@har.mrc.ac.uk.
authors.forename: Lydia
authors.initials: L
authors.lastname: Teboul
mesh_terms: D000483:Alleles; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004622:Embryo, Mammalian; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D016678:Genome; D060005:Genotyping Techniques; D054643:INDEL Mutation; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D017354:Point Mutation; D011786:Quality Control; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair; D015053:Zygote / Q000166:cytology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; Cas9; Genome editing; Genotyping; Mouse; Mutant; QC; Screening