A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells.
Sci Rep
; 10(1): 635, 2020 01 20.
Article
em En
| MEDLINE
| ID: mdl-31959800
ABSTRACT
CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene 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 and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained 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 the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results 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 can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Elementos de DNA Transponíveis
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RNA Guia de Cinetoplastídeos
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Técnicas de Transferência de Genes
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Células-Tronco Pluripotentes
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Sistemas CRISPR-Cas
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Vetores Genéticos
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos