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Genome Editing of Mammalian Cells Using CRISPR-Cas: From In Silico Designing to In-Culture Validation

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CRISPR-Cas Methods

Part of the book series: Springer Protocols Handbooks ((SPH))

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Abstract

The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease system has dramatically changed the field of cell and molecular biology. Any genomic sequence adjacent to a protospacer adjacent motif (PAM) site can potentially be edited by customizing the guide RNA (gRNA) that direct the Cas nuclease. Such versatility and flexibility have made the CRISPR-Cas system almost a default platform for genome editing nowadays. This system has gained widespread use also due to lower toxicity and simplicity of construction. Here, we describe a comprehensive protocol for gene deletion (knock-out) and tagging (knock-in) in mammalian cells, taking specific examples of Cas endonuclease systems and cell lines. However, the method can be adapted to edit genome employing other Cas plasmid systems as well as different human cell lines in a lab with standard cell and molecular biology facilities.

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References

  1. Dana H, Chalbatani GM, Mahmoodzadeh H et al (2017) Molecular mechanisms and biological functions of siRNA. Int J Biomed Sci 13:48–57

    PubMed  PubMed Central  Google Scholar 

  2. Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785. https://doi.org/10.1038/nbt1319

    Article  CAS  PubMed  Google Scholar 

  3. Moscou MJ, Bogdanove AJ (2009) A simple Cipher Governs DNA recognition by TAL effectors. Science 326(80):1501–1501. https://doi.org/10.1126/science.1178817

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339(80):823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Uddin B, Chen N-P, Panic M, Schiebel E (2015) Genome editing through large insertion leads to the skipping of targeted exon. BMC Genomics 16:1082. https://doi.org/10.1186/s12864-015-2284-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170. https://doi.org/10.1126/science.1179555

    Article  CAS  PubMed  Google Scholar 

  10. Ding Q, Regan SN, Xia Y et al (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394. https://doi.org/10.1016/J.STEM.2013.03.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fu Y, Sander JD, Reyon D et al (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279–284. https://doi.org/10.1038/nbt.2808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Slaymaker IM, Gao L, Zetsche B et al (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351(80):84–88. https://doi.org/10.1126/science.aad5227

    Article  CAS  PubMed  Google Scholar 

  13. Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Stewart SA, Dykxhoorn DM, Palliser D et al (2003) Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9:493–501

    Article  CAS  Google Scholar 

  15. Kulinski J, Besack D, Oleykowski CA et al (2000) CEL I Enzymatic mutation detection assay. Biotechniques 29:44–48. https://doi.org/10.2144/00291bm07

    Article  CAS  PubMed  Google Scholar 

  16. Weiss M (2016) Characterizing the functions of human CDC14A and CDC14B by CRISPR Cas9 mediated gene deletion approach. Heidelberg University, Heidelberg

    Google Scholar 

  17. Uddin B (2018) Linking dephosphorylation to cellular events: functional analysis of human CDC14 (hCDC14) phosphatases. Heidelberg University, Heidelberg

    Google Scholar 

  18. Fueller J, Meurer M, Herbst K et al (2018) CRISPR/Cas12a-assisted PCR tagging of mammalian genes. bioRxiv:1–31. https://doi.org/10.1101/473876

  19. Wang Y, Prosen DE, Mei L et al (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32:1197–1207. https://doi.org/10.1093/nar/gkh271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Uddin B, Partscht P, Chen N-P et al (2018) The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation. EMBO Rep:e46544. https://doi.org/10.15252/embr.201846544

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Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Jahangirnagar University, Dhaka, Bangladesh

    Borhan Uddin & Taslima Nahar

  2. Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Allianz, Heidelberg, Germany

    Patrick Partscht

  3. Heidelberg Biosciences International Graduate School (HBIGS), Universität Heidelberg, Heidelberg, Germany

    Patrick Partscht

Authors
  1. Borhan Uddin
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  2. Patrick Partscht
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  3. Taslima Nahar
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Corresponding author

Correspondence to Borhan Uddin .

Editor information

Editors and Affiliations

  1. Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

    M. Tofazzal Islam

  2. Department of Aquatic and Crop Resource Development, National Research Council of Canada, SASKATOON, SK, Canada

    Pankaj K. Bhowmik

  3. Crop Improvement Division, ICAR-National Rice Research Institute, Cuttack, Odisha, India

    Kutubuddin A. Molla

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Uddin, B., Partscht, P., Nahar, T. (2020). Genome Editing of Mammalian Cells Using CRISPR-Cas: From In Silico Designing to In-Culture Validation. In: Islam, M.T., Bhowmik, P.K., Molla, K.A. (eds) CRISPR-Cas Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0616-2_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0616-2_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0615-5

  • Online ISBN: 978-1-0716-0616-2

  • eBook Packages: Springer Protocols

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