title: Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote.
abstract: Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.
authors.affiliation: Institut Pasteur, CNRS, Unité de Génétique Fonctionnelle de la Souris, UMR 3738, Department of Developmental &Stem Cell Biology, 25 rue du docteur Roux, F-75015 Paris.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Raveux
authors.affiliation: Institut Pasteur, CNRS, Unité de Génétique Fonctionnelle de la Souris, UMR 3738, Department of Developmental &Stem Cell Biology, 25 rue du docteur Roux, F-75015 Paris.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Vandormael-Pournin
authors.affiliation: Institut Pasteur, CNRS, Unité de Génétique Fonctionnelle de la Souris, UMR 3738, Department of Developmental &Stem Cell Biology, 25 rue du docteur Roux, F-75015 Paris.
authors.forename: Michel
authors.initials: M
authors.lastname: Cohen-Tannoudji
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003600:Cytosol / Q000378:metabolism; D004622:Embryo, Mammalian; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D015053:Zygote / Q000166:cytology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; C510343:notchless protein, mouse; D049452:Green Fluorescent Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases