Efficient Prime Editing in Two-cell Mouse Embryos Using PEmbryo

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2024 Feb 6

PMID 38321114

Authors

Rebecca P Kim-Yip

Ryan McNulty

Bradley Joyce

Antonio Mollica

Peter J Chen

Purnima Ravisankar

Benjamin K Law

David R Liu

Jared E Toettcher

Evgueni A Ivakine

Eszter Posfai

Britt Adamson

Affiliations

Soon will be listed here.

Abstract

Using transient inhibition of DNA mismatch repair during a permissive stage of development, we demonstrate highly efficient prime editing of mouse embryos with few unwanted, local byproducts (average 58% precise edit frequency, 0.5% on-target error frequency across 13 substitution edits at 8 sites), enabling same-generation phenotyping of founders. Whole-genome sequencing reveals that mismatch repair inhibition increases off-target indels at low-complexity regions in the genome without any obvious phenotype in mice.

Citing Articles

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.

Cirincione A, Simpson D, Yan W, McNulty R, Ravisankar P, Solley S Nat Methods. 2024; 22(1):92-101.

PMID: 39562753 PMC: 11725502. DOI: 10.1038/s41592-024-02502-4.

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

Liu X, Gu D, Zhang Y, Jiang Y, Xiao Z, Xu R Genome Biol. 2024; 25(1):131.

PMID: 38773623 PMC: 11110357. DOI: 10.1186/s13059-024-03282-y.

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.

Cirincione A, Simpson D, Ravisankar P, Solley S, Yan J, Singh M bioRxiv. 2024; .

PMID: 38585933 PMC: 10996517. DOI: 10.1101/2024.03.25.585978.

Efficient prime editing in two-cell mouse embryos using PEmbryo.

Kim-Yip R, McNulty R, Joyce B, Mollica A, Chen P, Ravisankar P Nat Biotechnol. 2024; 42(12):1822-1830.

PMID: 38321114 PMC: 11631759. DOI: 10.1038/s41587-023-02106-x.

References

Guo R, Fang X, Mao H, Sun B, Zhou J, An Y . A Novel Missense Variant of Caused Atypical Synpolydactyly by Impairing the Downstream Gene Expression and Literature Review for Genotype-Phenotype Correlations. Front Genet. 2021; 12:731278. PMC: 8579070. DOI: 10.3389/fgene.2021.731278. View

Teboul L, Murray S, Nolan P . Phenotyping first-generation genome editing mutants: a new standard?. Mamm Genome. 2017; 28(7-8):377-382. PMC: 5569115. DOI: 10.1007/s00335-017-9711-x. View

Chen P, Hussmann J, Yan J, Knipping F, Ravisankar P, Chen P . Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021; 184(22):5635-5652.e29. PMC: 8584034. DOI: 10.1016/j.cell.2021.09.018. View

Liang P, Sun H, Sun Y, Zhang X, Xie X, Zhang J . Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell. 2017; 8(8):601-611. PMC: 5546933. DOI: 10.1007/s13238-017-0418-2. View

Bae S, Park J, Kim J . Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30(10):1473-5. PMC: 4016707. DOI: 10.1093/bioinformatics/btu048. View

Gu B, Gertsenstein M, Posfai E . Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos. Methods Mol Biol. 2019; 2066:89-100. DOI: 10.1007/978-1-4939-9837-1_7. View

Meier B, Volkova N, Hong Y, Schofield P, Campbell P, Gerstung M . Mutational signatures of DNA mismatch repair deficiency in and human cancers. Genome Res. 2018; 28(5):666-675. PMC: 5932607. DOI: 10.1101/gr.226845.117. View

Clark J, Dinsmore C, Soriano P . A most formidable arsenal: genetic technologies for building a better mouse. Genes Dev. 2020; 34(19-20):1256-1286. PMC: 7528699. DOI: 10.1101/gad.342089.120. View

Lin J, Liu X, Lu Z, Huang S, Wu S, Yu W . Modeling a cataract disorder in mice with prime editing. Mol Ther Nucleic Acids. 2021; 25:494-501. PMC: 8463286. DOI: 10.1016/j.omtn.2021.06.020. View

10.

Johnson K, Sweet H, Donahue L, Bronson R, Davisson M . A new spontaneous mouse mutation of Hoxd13 with a polyalanine expansion and phenotype similar to human synpolydactyly. Hum Mol Genet. 1998; 7(6):1033-8. DOI: 10.1093/hmg/7.6.1033. View

11.

Adikusuma F, Piltz S, Corbett M, Turvey M, McColl S, Helbig K . Large deletions induced by Cas9 cleavage. Nature. 2018; 560(7717):E8-E9. DOI: 10.1038/s41586-018-0380-z. View

12.

Dai L, Liu D, Song M, Xu X, Xiong G, Yang K . Mutations in the homeodomain of HOXD13 cause syndactyly type 1-c in two Chinese families. PLoS One. 2014; 9(5):e96192. PMC: 4006867. DOI: 10.1371/journal.pone.0096192. View

13.

Modzelewski A, Chen S, Willis B, Lloyd K, Wood J, He L . Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc. 2018; 13(6):1253-1274. PMC: 6296855. DOI: 10.1038/nprot.2018.012. View

14.

Gu B, Posfai E, Rossant J . Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol. 2018; 36(7):632-637. DOI: 10.1038/nbt.4166. View

15.

Petri K, Zhang W, Ma J, Schmidts A, Lee H, Horng J . CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat Biotechnol. 2021; 40(2):189-193. PMC: 8553808. DOI: 10.1038/s41587-021-00901-y. View

16.

Park S, Jeong T, Shin S, Yoon D, Lim S, Kim S . Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor. Genome Biol. 2021; 22(1):170. PMC: 8173820. DOI: 10.1186/s13059-021-02389-w. View

17.

Schofield M, Hsieh P . DNA mismatch repair: molecular mechanisms and biological function. Annu Rev Microbiol. 2003; 57:579-608. DOI: 10.1146/annurev.micro.57.030502.090847. View

18.

Choi J, Chen W, Suiter C, Lee C, Chardon F, Yang W . Precise genomic deletions using paired prime editing. Nat Biotechnol. 2021; 40(2):218-226. PMC: 8847327. DOI: 10.1038/s41587-021-01025-z. View

19.

Ciemerych M, Sicinski P . Cell cycle in mouse development. Oncogene. 2005; 24(17):2877-98. DOI: 10.1038/sj.onc.1208608. View

20.

Liu P, Liang S, Zheng C, Mintzer E, Zhao Y, Ponnienselvan K . Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat Commun. 2021; 12(1):2121. PMC: 8035190. DOI: 10.1038/s41467-021-22295-w. View