Generation of an Inducible DCas9-SAM Human PSC Line for Endogenous Gene Activation

Overview

Journal Front Cell Dev Biol

Specialty Cell Biology

Date 2024 Dec 16

PMID 39676795

Authors

Paolo Petazzi

Francisco Gutierrez-Aguera

Heleia Roca-Ho

Julio Castano

Clara Bueno

Niuska Alvarez

Lesley M Forrester

Ana Sevilla

Antonella Fidanza

Pablo Menendez

Affiliations

Soon will be listed here.

Abstract

The CRISPR/Cas9 system has transformed genome editing by enabling precise modifications for diverse applications. Recent advancements, including base editing and prime editing, have expanded its utility beyond conventional gene knock-out and knock-in strategies. Additionally, several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains have been developed to modulate endogenous gene expression when directed to their regulatory regions by specific single-guide RNAs. Here, we report the development of the H9 human pluripotent stem cell (hPSC) line expressing an inducible dCas9-SAM activator (H9-iCas9.SAM), designed to activate transcription of endogenous genes. The H9-iCas9.SAM cells were generated through targeted integration of an inducible CRISPR/Cas9-based gene activator cassette into the AAVS1 "safe-harbour" locus. Molecular analyses confirmed precise and specific integration, ensuring minimal off-target effects. Functional characterization revealed that H9-iCas9.SAM cells retain pluripotency and display inducible endogenous gene activation upon doxycycline treatment. The versatility of H9-iCas9.SAM cells was demonstrated in directed differentiation assays, yielding neural stem cells (ectoderm), hematopoietic progenitor cells (mesoderm), and hepatocytes (endoderm). This underscores their potential in developmental biology studies and cell therapy applications. The engineered H9-iCas9.SAM line provides a robust platform for investigating gene function and advancing next-generation cell-based therapies.

References

Yeo N, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D . An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods. 2018; 15(8):611-616. PMC: 6129399. DOI: 10.1038/s41592-018-0048-5. View

Jiang L, Liang J, Huang W, Ma J, Park K, Wu Z . CRISPR activation of endogenous genes reprograms fibroblasts into cardiovascular progenitor cells for myocardial infarction therapy. Mol Ther. 2021; 30(1):54-74. PMC: 8753567. DOI: 10.1016/j.ymthe.2021.10.015. View

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

Petazzi P, Torres-Ruiz R, Fidanza A, Roca-Ho H, Gutierrez-Aguera F, Castano J . Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells. Mol Ther Nucleic Acids. 2020; 20:196-204. PMC: 7068053. DOI: 10.1016/j.omtn.2020.02.009. View

Bueno C, Montes R, Melen G, Ramos-Mejia V, Real P, Ayllon V . A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification. Cell Res. 2012; 22(6):986-1002. PMC: 3367544. DOI: 10.1038/cr.2012.4. View

Kowalski K, Brzoska E, Ciemerych M . The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation. Stem Cell Res. 2019; 41:101636. DOI: 10.1016/j.scr.2019.101636. View

Menendez P, Bueno C, Wang L . Human embryonic stem cells: A journey beyond cell replacement therapies. Cytotherapy. 2006; 8(6):530-41. DOI: 10.1080/14653240601026654. View

Madhavan M, Nevin Z, Shick H, Garrison E, Clarkson-Paredes C, Karl M . Induction of myelinating oligodendrocytes in human cortical spheroids. Nat Methods. 2018; 15(9):700-706. PMC: 6508550. DOI: 10.1038/s41592-018-0081-4. View

Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung J . CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10(10):977-9. PMC: 3794058. DOI: 10.1038/nmeth.2598. View

10.

Fidanza A, Lopez-Yrigoyen M, Romano N, Jones R, Taylor A, Forrester L . An all-in-one UniSam vector system for efficient gene activation. Sci Rep. 2017; 7(1):6394. PMC: 5526871. DOI: 10.1038/s41598-017-06468-6. View

11.

Komor A, Kim Y, Packer M, Zuris J, Liu D . Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016; 533(7603):420-4. PMC: 4873371. DOI: 10.1038/nature17946. View

12.

Li J, Li R, Bai X, Zhang W, Nie Y, Hu S . Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3. Chin Med J (Engl). 2024; 137(11):1351-1359. PMC: 11191006. DOI: 10.1097/CM9.0000000000003088. View

13.

Castano J, Bueno C, Jimenez-Delgado S, Roca-Ho H, Fraga M, Fernandez A . Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the "safe harbor" AAVS1 locus. Stem Cell Res. 2017; 21:137-140. PMC: 5446316. DOI: 10.1016/j.scr.2017.04.011. View

14.

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

15.

Thomson J, Itskovitz-Eldor J, Shapiro S, Waknitz M, Swiergiel J, Marshall V . Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282(5391):1145-7. DOI: 10.1126/science.282.5391.1145. View

16.

Mandegar M, Huebsch N, Frolov E, Shin E, Truong A, Olvera M . CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell. 2016; 18(4):541-53. PMC: 4830697. DOI: 10.1016/j.stem.2016.01.022. View

17.

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

18.

Woods L, Ali F, Gomez R, Chernukhin I, Marcos D, Parkinson L . Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation. BMC Genomics. 2022; 23(1):255. PMC: 8977041. DOI: 10.1186/s12864-022-08495-8. View

19.

Matsuda-Ito K, Matsuda T, Nakashima K . Expression level of the reprogramming factor NeuroD1 is critical for neuronal conversion efficiency from different cell types. Sci Rep. 2022; 12(1):17980. PMC: 9606360. DOI: 10.1038/s41598-022-22802-z. View

20.

Liu X, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S . Editing DNA Methylation in the Mammalian Genome. Cell. 2016; 167(1):233-247.e17. PMC: 5062609. DOI: 10.1016/j.cell.2016.08.056. View