One-step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-mediated Genome Engineering

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2013 Sep 3

PMID 23992847

Citations 847

Authors

Hui Yang

Haoyi Wang

Chikdu S Shivalila

Albert W Cheng

Linyu Shi

Rudolf Jaenisch

Affiliations

Soon will be listed here.

Abstract

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Citing Articles

Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease.

Gertsenstein M, Lintott L, Nutter L Curr Protoc. 2025; 5(2):e70109.

PMID: 39999224 PMC: 11856344. DOI: 10.1002/cpz1.70109.

Gene expression of psychiatric disorder-related kinesin superfamily proteins (Kifs) is potentiated in alternatively activated primary cultured microglia.

Iwata S, Hyugaji M, Soga Y, Morikawa M, Sasaki T, Takei Y BMC Res Notes. 2025; 18(1):44.

PMID: 39885501 PMC: 11783738. DOI: 10.1186/s13104-024-07078-y.

Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.

Hazel K, Singh D, He S, Guertin Z, Husser M, Helfield B Mol Ther. 2025; 33(3):986-996.

PMID: 39797397 PMC: 11897754. DOI: 10.1016/j.ymthe.2025.01.013.

Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation.

Sakurai T, Takei N, Wei Y, Hayashi M, Kamiyoshi A, Kawate H Sci Rep. 2024; 14(1):30347.

PMID: 39639057 PMC: 11621336. DOI: 10.1038/s41598-024-81198-0.

Twenty-first century mouse genetics is again at an inflection point.

Fang Z, Peltz G Lab Anim (NY). 2024; 54(1):9-15.

PMID: 39592878 PMC: 11695262. DOI: 10.1038/s41684-024-01491-3.

References

Sung Y, Baek I, Kim D, Jeon J, Lee J, Lee K . Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol. 2013; 31(1):23-4. DOI: 10.1038/nbt.2477. View

Hwang W, Fu Y, Reyon D, Maeder M, Tsai S, Sander J . Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013; 31(3):227-9. PMC: 3686313. DOI: 10.1038/nbt.2501. View

Wiedenheft B, Sternberg S, Doudna J . RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482(7385):331-8. DOI: 10.1038/nature10886. View

Hsu P, Scott D, Weinstein J, Ran F, Konermann S, Agarwala V . DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31(9):827-32. PMC: 3969858. DOI: 10.1038/nbt.2647. View

Wefers B, Meyer M, Ortiz O, de Angelis M, Hansen J, Wurst W . Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A. 2013; 110(10):3782-7. PMC: 3593923. DOI: 10.1073/pnas.1218721110. View

Capecchi M . Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 2005; 6(6):507-12. DOI: 10.1038/nrg1619. View

Gilbert L, Larson M, Morsut L, Liu Z, Brar G, Torres S . CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2):442-51. PMC: 3770145. DOI: 10.1016/j.cell.2013.06.044. View

Faddah D, Wang H, Cheng A, Katz Y, Buganim Y, Jaenisch R . Single-cell analysis reveals that expression of nanog is biallelic and equally variable as that of other pluripotency factors in mouse ESCs. Cell Stem Cell. 2013; 13(1):23-9. PMC: 4035816. DOI: 10.1016/j.stem.2013.04.019. View

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

10.

Fu Y, Foden J, Khayter C, Maeder M, Reyon D, Joung J . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9):822-6. PMC: 3773023. DOI: 10.1038/nbt.2623. View

11.

Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J . RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMC: 3712628. DOI: 10.1126/science.1232033. View

12.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View

13.

Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J . RNA-programmed genome editing in human cells. Elife. 2013; 2:e00471. PMC: 3557905. DOI: 10.7554/eLife.00471. View

14.

Lengner C, Camargo F, Hochedlinger K, Welstead G, Zaidi S, Gokhale S . Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell. 2007; 1(4):403-15. PMC: 2151746. DOI: 10.1016/j.stem.2007.07.020. View

15.

Guschin D, Waite A, Katibah G, Miller J, Holmes M, Rebar E . A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol. 2010; 649:247-56. DOI: 10.1007/978-1-60761-753-2_15. View

16.

Geurts A, Cost G, Freyvert Y, Zeitler B, Miller J, Choi V . Knockout rats via embryo microinjection of zinc-finger nucleases. Science. 2009; 325(5939):433. PMC: 2831805. DOI: 10.1126/science.1172447. View

17.

Brown A, Fisher D, Kouranova E, McCoy A, Forbes K, Wu Y . Whole-rat conditional gene knockout via genome editing. Nat Methods. 2013; 10(7):638-40. DOI: 10.1038/nmeth.2516. View

18.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View

19.

Wang H, Yang H, Shivalila C, Dawlaty M, Cheng A, Zhang F . One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013; 153(4):910-8. PMC: 3969854. DOI: 10.1016/j.cell.2013.04.025. View

20.

Horvath P, Barrangou R . CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010; 327(5962):167-70. DOI: 10.1126/science.1179555. View