Abstract
CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sakuma T, Woltjen K (2014) Nuclease-mediated genome editing: at the front-line of functional genomics technology. Dev Growth Differ 56:2–13
Sakuma T, Yamamoto T (2015) CRISPR/Cas9: the leading edge of genome editing technology. In: Yamamoto T (ed) Targeted genome editing using site-specific nucleases: ZFNs, TALENs, and the CRISPR/Cas9 system. Springer, Japan, pp 25–41
Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T (2014) Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep 4:5400
Nakagawa Y, Sakuma T, Sakamoto T, Ohmuraya M, Nakagata N, Yamamoto T (2015) Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol 15:33
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647
Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82
Sakuma T, Hosoi S, Woltjen K, Suzuki K, Kashiwagi K, Wada H, Ochiai H, Miyamoto T, Kawai N, Sasakura Y, Matsuura S, Okada Y, Kawahara A, Hayashi S, Yamamoto T (2013) Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18:315–326
Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, Suzuki K, Miyamoto T, Sakamoto N, Matsuura S, Yamamoto T (2013) Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci Rep 3:3379
Sakuma T, Yamamoto T (2016) Engineering customized TALENs using the platinum gate TALEN kit. Methods Mol Biol 1338:61–70
Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560
Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H, Yamamoto T, Kawahara A (2015) Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci Rep 5:8841
Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T (2016) MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 11:118–133
Sakuma T (2015) Front-line of genome editing technology for animal cell engineering. BMC Proc 9(Suppl 9):O1
Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569–576
Guilinger JP, Thompson DB, Liu DR (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol 32:577–582
Hara S, Tamano M, Yamashita S, Kato T, Saito T, Sakuma T, Yamamoto T, Inui M, Takada S (2015) Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9. Sci Rep 5:11221
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832
Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–1389
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826
Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, Ye Z (2014) Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15:12–13
Feng Y, Sassi S, Shen JK, Yang X, Gao Y, Osaka E, Zhang J, Yang S, Yang C, Mankin HJ, Hornicek FJ, Duan Z (2015) Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system. J Orthop Res 33:199–207
Ebina H, Kanemura Y, Misawa N, Sakuma T, Kobayashi T, Yamamoto T, Koyanagi Y (2015) A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector. PLoS One 10:e0120047
Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53
Li HL, Gee P, Ishida K, Hotta A (2016) Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system. Methods. doi:10.1016/j.ymeth.2015.10.015
Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Long X, Hatani T, Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K (2016) Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. Methods. doi:10.1016/j.ymeth.2015.12.012
Matsubara Y, Chiba T, Kashimada K, Morio T, Takada S, Mizutani S, Asahara H (2014) Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus. Sci Rep 4:5043
Rao S, Fujimura T, Matsunari H, Sakuma T, Nakano K, Watanabe M, Asano Y, Kitagawa E, Yamamoto T, Nagashima H (2016) Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets. Mol Reprod Dev. doi:10.1002/mrd.22591
Kaneko T, Mashimo T (2015) Simple genome editing of rodent intact embryos by electroporation. PLoS One 10:e0142755
Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382
Ota S, Hisano Y, Muraki M, Hoshijima K, Dahlem TJ, Grunwald DJ, Okada Y, Kawahara A (2013) Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18:450–458
Nakagawa Y, Yamamoto T, Suzuki K, Araki K, Takeda N, Ohmuraya M, Sakuma T (2014) Screening methods to identify TALEN-mediated knockout mice. Exp Anim 63:79–84
Suzuki KT, Isoyama Y, Kashiwagi K, Sakuma T, Ochiai H, Sakamoto N, Furuno N, Kashiwagi A, Yamamoto T (2013) High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos. Biol Open 2:448–452
Dahlem TJ, Hoshijima K, Jurynec MJ, Gunther D, Starker CG, Locke AS, Weis AM, Voytas DF, Grunwald DJ (2012) Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 8:e1002861
Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256
Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G (2014) COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol Ther Nucleic Acids 3:e214
Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473–7485
Acknowledgments
This work was supported by the Uehara Memorial Foundation (to T.S.), the Japan Society for the Promotion of Science (16K18478 to T.S. and 26290070 to T.Y.), and the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) to T.Y. The authors wish to express their thanks to Prof. Kazuaki Chayama, Department of Gastroenterology and Metabolism, Applied Life Science, Institute of Biomedical and Health Science, Hiroshima University, for his helpful suggestions concerning HepG2 cell culture.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sakuma, T., Sakamoto, T., Yamamoto, T. (2017). All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6472-7_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6470-3
Online ISBN: 978-1-4939-6472-7
eBook Packages: Springer Protocols