title: Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.
abstract: The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.
authors.affiliation: From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705.
authors.forename: Sean
authors.initials: S
authors.lastname: Chen
authors.affiliation: From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lee
authors.affiliation: From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705.
authors.forename: Angus Yiu-Fai
authors.initials: AY
authors.lastname: Lee
authors.affiliation: From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705 modzelewski.andrew@berkeley.edu.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Modzelewski
authors.affiliation: From the Division of Cellular and Developmental Biology, MCB Department, University of California at Berkeley, Berkeley, California 94705 lhe@berkeley.edu.
authors.forename: Lin
authors.initials: L
authors.lastname: He
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D018274:Electroporation / Q000379:methods*; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000008:administration & dosage* / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics*; D015053:Zygote / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012261:Ribonucleoproteins
keywords: CRISPR, Cas9, Genome Editing, Electroporation, RNP; gene knockout; genetics; mouse; ribonuclear protein (RNP); tyrosinase