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 language | English (US) |
|---|---|
| Pages (from-to) | 1253-1274 |
| Number of pages | 22 |
| Journal | Nature Protocols |
| Volume | 13 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 1 2018 |
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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 journal › Article
}
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.
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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 -