Targeted genome engineering using designer nucleases: State of the art and practical guidance for application in human pluripotent stem cells

ABSTRACTCRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional genomic studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design new fluorescently labeledpiggyBac(PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target theTCF4gene and conduct a rigorous assessment of expression levels of the dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system would facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

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P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

10.1101/168443 ◽

2017 ◽

Cited By ~ 2

Author(s):

Robert J. Ihry ◽

Kathleen A. Worringer ◽

Max R. Salick ◽

Elizabeth Frias ◽

Daniel Ho ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Genome Engineering ◽

High Efficiency ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Double Strand Breaks ◽

Toxic Response ◽

Strand Breaks ◽

Genome Wide

SUMMARYCRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response istp53-dependent and the toxic effect oftp53severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor fortp53function, especially in hPSCs which spontaneously acquiretp53mutations.

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Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cells ◽

10.3390/cells9051312 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1312 ◽

Cited By ~ 2

Author(s):

Joffrey Mianné ◽

Chloé Bourguignon ◽

Chloé Nguyen Van ◽

Mathieu Fieldès ◽

Amel Nasri ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Genome Engineering ◽

Disease Modeling ◽

Mutant Cell ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Wide Range

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

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