A Chemical Approach Facilitates CRISPRa-only Human IPSC Generation and Minimizes the Number of Targeted Loci Required

Overview

Journal Future Sci OA

Specialty Biotechnology

Date 2024 May 31

PMID 38817352

Authors

Ramzey Abujarour

Jason Dinella

Mochtar Pribadi

Lauren K Fong

Matthew Denholtz

Alma Gutierrez

Matt Haynes

Enaaya Mahmood

Tom T Lee

Sheng Ding

Bahram Valamehr

Affiliations

Soon will be listed here.

Abstract

We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by and . CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

References

Buganim Y, Faddah D, Cheng A, Itskovich E, Markoulaki S, Ganz K . Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell. 2012; 150(6):1209-22. PMC: 3457656. DOI: 10.1016/j.cell.2012.08.023. View

Chavez A, Scheiman J, Vora S, Pruitt B, Tuttle M, Iyer E . Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015; 12(4):326-8. PMC: 4393883. DOI: 10.1038/nmeth.3312. View

Xiong K, Zhou Y, Blichfeld K, Hyttel P, Bolund L, Freude K . RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts. Cell Reprogram. 2017; 19(3):189-198. DOI: 10.1089/cell.2017.0006. View

Chavez A, Tuttle M, Pruitt B, Ewen-Campen B, Chari R, Ter-Ovanesyan D . Comparison of Cas9 activators in multiple species. Nat Methods. 2016; 13(7):563-567. PMC: 4927356. DOI: 10.1038/nmeth.3871. View

Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T . Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Reports. 2014; 2(3):366-81. PMC: 3964282. DOI: 10.1016/j.stemcr.2014.01.014. View

Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi D, Kano Y . Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell. 2011; 8(6):633-8. DOI: 10.1016/j.stem.2011.05.001. View

Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, Shoemaker D . A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep. 2012; 2:213. PMC: 3252544. DOI: 10.1038/srep00213. View

Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti E . Human pluripotent reprogramming with CRISPR activators. Nat Commun. 2018; 9(1):2643. PMC: 6035213. DOI: 10.1038/s41467-018-05067-x. View

Abujarour R, Valamehr B, Robinson M, Rezner B, Vranceanu F, Flynn P . Optimized surface markers for the prospective isolation of high-quality hiPSCs using flow cytometry selection. Sci Rep. 2013; 3:1179. PMC: 3560358. DOI: 10.1038/srep01179. View

10.

Tanenbaum M, Gilbert L, Qi L, Weissman J, Vale R . A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014; 159(3):635-46. PMC: 4252608. DOI: 10.1016/j.cell.2014.09.039. View

11.

Tang S, Xie M, Cao N, Ding S . Patient-Specific Induced Pluripotent Stem Cells for Disease Modeling and Phenotypic Drug Discovery. J Med Chem. 2015; 59(1):2-15. DOI: 10.1021/acs.jmedchem.5b00789. View

12.

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-72. DOI: 10.1016/j.cell.2007.11.019. View

13.

Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T . Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation. Stem Cell Reports. 2015; 5(3):448-59. PMC: 4618656. DOI: 10.1016/j.stemcr.2015.08.001. View

14.

Yu J, Vodyanik M, Smuga-Otto K, Antosiewicz-Bourget J, Frane J, Tian S . Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858):1917-20. DOI: 10.1126/science.1151526. View

15.

Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A . An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2012; 31(3):458-66. DOI: 10.1002/stem.1293. View

16.

Konermann S, Brigham M, Trevino A, Joung J, Abudayyeh O, Barcena C . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014; 517(7536):583-8. PMC: 4420636. DOI: 10.1038/nature14136. View

17.

Subramanyam D, Lamouille S, Judson R, Liu J, Bucay N, Derynck R . Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat Biotechnol. 2011; 29(5):443-8. PMC: 3685579. DOI: 10.1038/nbt.1862. View

18.

Dominguez A, Lim W, Qi L . Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2015; 17(1):5-15. PMC: 4922510. DOI: 10.1038/nrm.2015.2. View

19.

Liu P, Chen M, Liu Y, Qi L, Ding S . CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency. Cell Stem Cell. 2018; 22(2):252-261.e4. DOI: 10.1016/j.stem.2017.12.001. View

20.

Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R . A chemical platform for improved induction of human iPSCs. Nat Methods. 2009; 6(11):805-8. PMC: 3724527. DOI: 10.1038/nmeth.1393. View