Top

Published in:

2015 | Online First | Chapter

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool

Authors : Rahel Wettstein, Maxime Bodak, Constance Ciaudo, Ph.D.

Published in: Methods in Molecular Biology™

Publisher: Springer New York

Activate our intelligent search to find suitable subject content or patents.

search-config

AI-assisted search

Off

Abstract

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.

Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

Martello G, Smith A (2014) The nature of embryonic stem cells. Annu Rev Cell Dev Biol 30:647–675. doi:10.1146/annurev-cellbio-100913-013116 CrossRefPubMed

Boyer LA, Mathur D, Jaenisch R (2006) Molecular control of pluripotency. Curr Opin Genet Dev 16:455–462. doi:10.1016/j.gde.2006.08.009 CrossRefPubMed

Beyer TA, Narimatsu M, Weiss A et al (2013) The TGFβ superfamily in stem cell biology and early mammalian embryonic development. Biochim Biophys Acta 1830:2268–2279. doi:10.1016/j.bbagen.2012.08.025 CrossRefPubMed

Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680. doi:10.1016/j.cell.2008.02.008 CrossRefPubMed

Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278. doi:10.1016/j.cell.2014.05.010 PubMedCentralCrossRefPubMed

Ishino Y, Shinagawa H, Makino K et al (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433PubMedCentralPubMed

Jansen R, Embden JDA, Van Gaastra W, Schouls LM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43:1565–1575. doi:10.1046/j.1365-2958.2002.02839.x CrossRefPubMed

Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. doi:10.1126/science.1138140 CrossRefPubMed

Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. doi:10.1126/science.1232033.RNA-Guided PubMedCentralCrossRefPubMed

10.

Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi:10.1126/science.1231143 PubMedCentralCrossRefPubMed

11.

Deltcheva E, Chylinski K, Sharma CM, Gonzales K (2011) CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III. Nature 471:602–607. doi:10.1038/nature09886.CRISPR PubMedCentralCrossRefPubMed

12.

Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. doi:10.1126/science.1225829 CrossRefPubMed

13.

Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740. doi:10.1099/mic. 0.023960-0 CrossRefPubMed

14.

Deveau H, Barrangou R, Garneau JE et al (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190:1390–1400. doi:10.1128/JB.01412-07 PubMedCentralCrossRefPubMed

15.

Semenova E, Jore MM, Datsenko KA et al (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108:10098–10103. doi:10.1073/pnas.1104144108 PubMedCentralCrossRefPubMed

16.

Jinek M, Jiang F, Taylor DW et al (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343:1247997. doi:10.1126/science.1247997 PubMedCentralCrossRefPubMed

17.

Gilbert LA, Horlbeck MA, Adamson B et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–661. doi:10.1016/j.cell.2014.09.029 PubMedCentralCrossRefPubMed

18.

Kearns NA, Genga RMJ, Enuameh MS et al (2014) Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141:219–223. doi:10.1242/dev.103341 PubMedCentralCrossRefPubMed

19.

Tanenbaum ME, Gilbert LA, Qi LS et al (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–646. doi:10.1016/j.cell.2014.09.039 PubMedCentralCrossRefPubMed

20.

O‘Connell MR, Oakes BL, Sternberg SH et al (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. doi:10.1038/nature13769 PubMedCentral

21.

Heigwer F, Kerr G, Boutros M (2014) E-CRISP: fast CRISPR target site identification. Nat Methods 11:122–123. doi:10.1038/nmeth.2812 CrossRefPubMed

22.

Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. doi:10.1038/nbt.2647 PubMedCentralCrossRefPubMed

23.

Bae S, Park J, Kim J-S (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475. doi:10.1093/bioinformatics/btu048 PubMedCentralCrossRefPubMed

24.

Montague TG, Cruz JM, Gagnon JA et al (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:401–7. doi:10.1093/nar/gku410 CrossRef

25.

Xiao A, Cheng Z, Kong L et al (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180–1182. doi:10.1093/bioinformatics/btt764 CrossRef

26.

Koike-Yusa H, Li Y, Tan E-P et al (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32:267–273. doi:10.1038/nbt.2800 CrossRefPubMed

27.

Nora EP, Heard E (2011) Engineering genomic deletions and inversions in mouse ES cells using custom designed nucleases 1–15, Epigenesys website, protocol #62 (http://www.epigenesys.eu/images/stories/protocols/pdf/20130507072445_p62.pdf).

28.

Canver MC, Bauer DE, Dass A et al (2014) Characterization of genomic deletion efficiency mediated by CRISPR/Cas9 in mammalian cells. J Biol Chem. doi:10.1074/jbc.M114.564625 PubMedCentralPubMed

29.

Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308. doi:10.1038/nprot.2013.143 PubMedCentralCrossRefPubMed

30.

Marx V (2014) Gene editing: how to stay on-target with CRISPR. Nat Methods 11:1021–1026. doi:10.1038/nmeth.3108 CrossRef

31.

Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. doi:10.1186/1471-2105-13-134 PubMedCentralCrossRefPubMed

Title: Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool
Authors: Rahel Wettstein
Maxime Bodak
Constance Ciaudo, Ph.D.
Publisher: Springer New York
DOI: https://doi.org/10.1007/7651_2015_213

Springer Professional

Abstract

Please log in to get access to your license.