Efficient mouse genome engineering by CRISPR-EZ technology

Andrew J. Modzelewski, Sean Chen, Brandon J. Willis, Kevin C K Lloyd, Joshua Wood, Lin He

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

Original languageEnglish (US)
Pages (from-to)1253-1274
Number of pages22
JournalNature Protocols
Volume13
Issue number6
DOIs
StatePublished - Jun 1 2018

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Genes
Genome
Technology
Electroporation
Guide RNA
Ribonucleoproteins
Embryonic Structures
Throughput
Microinjections
INDEL Mutation
Zygote
Sequence Deletion
Inbred C57BL Mouse
Point Mutation
Knockout Mice
Exons
Pipelines
Economics
Research Personnel

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Efficient mouse genome engineering by CRISPR-EZ technology. / Modzelewski, Andrew J.; Chen, Sean; Willis, Brandon J.; Lloyd, Kevin C K; Wood, Joshua; He, Lin.

In: Nature Protocols, Vol. 13, No. 6, 01.06.2018, p. 1253-1274.

Research output: Contribution to journalArticle

Modzelewski, Andrew J. ; Chen, Sean ; Willis, Brandon J. ; Lloyd, Kevin C K ; Wood, Joshua ; He, Lin. / Efficient mouse genome engineering by CRISPR-EZ technology. In: Nature Protocols. 2018 ; Vol. 13, No. 6. pp. 1253-1274.
@article{524be4e48af747b29d2f3449d7b61e99,
title = "Efficient mouse genome engineering by CRISPR-EZ technology",
abstract = "CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100{\%} delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.",
author = "Modzelewski, {Andrew J.} and Sean Chen and Willis, {Brandon J.} and Lloyd, {Kevin C K} and Joshua Wood and Lin He",
year = "2018",
month = "6",
day = "1",
doi = "10.1038/nprot.2018.012",
language = "English (US)",
volume = "13",
pages = "1253--1274",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "6",

}

TY - JOUR

T1 - Efficient mouse genome engineering by CRISPR-EZ technology

AU - Modzelewski, Andrew J.

AU - Chen, Sean

AU - Willis, Brandon J.

AU - Lloyd, Kevin C K

AU - Wood, Joshua

AU - He, Lin

PY - 2018/6/1

Y1 - 2018/6/1

N2 - CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

AB - CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

UR - http://www.scopus.com/inward/record.url?scp=85047516059&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85047516059&partnerID=8YFLogxK

U2 - 10.1038/nprot.2018.012

DO - 10.1038/nprot.2018.012

M3 - Article

C2 - 29748649

AN - SCOPUS:85047516059

VL - 13

SP - 1253

EP - 1274

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 6

ER -