Targeted Mutagenesis in Human IPSCs Using CRISPR Genome-editing Tools

Overview

Journal Methods

Specialty Biochemistry

Date 2021 Jan 14

PMID 33444739

Citations 2

Authors

Yicheng Long

Thomas R Cech

Affiliations

Soon will be listed here.

Abstract

Mutagenesis studies have rapidly evolved in the era of CRISPR genome editing. Precise manipulation of genes in human induced pluripotent stem cells (iPSCs) allows biomedical researchers to study the physiological functions of individual genes during development. Furthermore, such genetic manipulation applied to patient-specific iPSCs allows disease modeling, drug screening and development of therapeutics. Although various genome-editing methods have been developed to introduce or remove mutations in human iPSCs, comprehensive strategic designs taking account of the potential side effects of CRISPR editing are needed. Here we present several novel and highly efficient strategies to introduce point mutations, insertions and deletions in human iPSCs, including step-by-step experimental protocols. These approaches involve the application of drug selection for effortless clone screening and the generation of a wild type control strain along with the mutant. We also present several examples of application of these strategies in human iPSCs and show that they are highly efficient and could be applied to other cell types.

Citing Articles

Evaluation of the RNA-dependence of PRC2 binding to chromatin in human pluripotent stem cells.

Long Y, Hwang T, Gooding A, Goodrich K, Hanson S, Vallery T bioRxiv. 2023; .

PMID: 37645830 PMC: 10462166. DOI: 10.1101/2023.08.17.553776.

Modulating DNA Repair Pathways to Diversify Genomic Alterations in Saccharomyces cerevisiae.

Wang Z, Lin Y, Dai Z, Wang Q Microbiol Spectr. 2022; 10(2):e0232621.

PMID: 35352941 PMC: 9045378. DOI: 10.1128/spectrum.02326-21.

References

Carroll D, Beumer K . Genome engineering with TALENs and ZFNs: repair pathways and donor design. Methods. 2014; 69(2):137-41. PMC: 4175112. DOI: 10.1016/j.ymeth.2014.03.026. View

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

Gasiunas G, Barrangou R, Horvath P, Siksnys V . Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012; 109(39):E2579-86. PMC: 3465414. DOI: 10.1073/pnas.1208507109. View

Li X, Li G, Fu J, Fu Y, Zhang L, Chen W . Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. Nucleic Acids Res. 2018; 46(19):10195-10215. PMC: 6212847. DOI: 10.1093/nar/gky804. View

Paix A, Folkmann A, Goldman D, Kulaga H, Grzelak M, Rasoloson D . Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. Proc Natl Acad Sci U S A. 2017; 114(50):E10745-E10754. PMC: 5740635. DOI: 10.1073/pnas.1711979114. View

Tian B, Hu J, Zhang H, Lutz C . A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res. 2005; 33(1):201-12. PMC: 546146. DOI: 10.1093/nar/gki158. View

Chatterjee P, Cheung Y, Liew C . Transfecting and nucleofecting human induced pluripotent stem cells. J Vis Exp. 2011; (56). PMC: 3227177. DOI: 10.3791/3110. View

Yang L, Guell M, Byrne S, Yang J, De Los Angeles A, Mali P . Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 2013; 41(19):9049-61. PMC: 3799423. DOI: 10.1093/nar/gkt555. View

Tuladhar R, Yeu Y, Tyler Piazza J, Tan Z, Clemenceau J, Wu X . CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat Commun. 2019; 10(1):4056. PMC: 6731291. DOI: 10.1038/s41467-019-12028-5. View

10.

Sekine R, Kawata T, Muramoto T . CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium. Sci Rep. 2018; 8(1):8471. PMC: 5981456. DOI: 10.1038/s41598-018-26756-z. View

11.

Gutschner T, Baas M, Diederichs S . Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases. Genome Res. 2011; 21(11):1944-54. PMC: 3205578. DOI: 10.1101/gr.122358.111. View

12.

Merkle F, Ghosh S, Kamitaki N, Mitchell J, Avior Y, Mello C . Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 2017; 545(7653):229-233. PMC: 5427175. DOI: 10.1038/nature22312. View

13.

Hockemeyer D, Jaenisch R . Gene targeting in human pluripotent cells. Cold Spring Harb Symp Quant Biol. 2011; 75:201-9. DOI: 10.1101/sqb.2010.75.021. View

14.

Schmidt J, Zaug A, Cech T . Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres. Cell. 2016; 166(5):1188-1197.e9. PMC: 5743434. DOI: 10.1016/j.cell.2016.07.033. View

15.

Proudfoot N, Brownlee G . 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976; 263(5574):211-4. DOI: 10.1038/263211a0. View

16.

Lin J, Musunuru K . Genome engineering tools for building cellular models of disease. FEBS J. 2016; 283(17):3222-31. PMC: 5881911. DOI: 10.1111/febs.13763. View

17.

Sheets M, Ogg S, Wickens M . Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic Acids Res. 1990; 18(19):5799-805. PMC: 332317. DOI: 10.1093/nar/18.19.5799. View

18.

Byrne S, Mali P, Church G . Genome editing in human stem cells. Methods Enzymol. 2014; 546:119-38. PMC: 4408990. DOI: 10.1016/B978-0-12-801185-0.00006-4. View

19.

Laird P, Zijderveld A, Linders K, Rudnicki M, Jaenisch R, Berns A . Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 1991; 19(15):4293. PMC: 328579. DOI: 10.1093/nar/19.15.4293. View

20.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View