Generation of induced pluripotent cells by CRISPR activation
Inventors
Assignees
Publication Number
US-12146166-B2
Publication Date
2024-11-19
Expiration Date
2038-12-27
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Abstract
The present Application is related to methods and compositions for reprogramming adult somatic cells into induced pluripotent stem cells by targeting and remodeling endogenous gene loci without relying on ectopic expression of transcription factors.
Core Innovation
The invention relates to methods and compositions for generating induced pluripotent stem cells (iPSCs) by targeting and remodeling endogenous gene loci in adult somatic cells using a CRISPR activation (CRISPRa) system without requiring ectopic expression of transcription factors. Specifically, the methods employ single guide RNAs (sgRNAs) directed to endogenous genes such as Oct4, Sox2, and others, combined with a CRISPR activation system comprising nuclease-deactivated Cas9 fused to transcriptional activators like VP64 or p300core. This targeted remodeling reactivates the pluripotency network within somatic cells, thereby inducing reprogramming to pluripotency.
The problem addressed is the limitation and risks associated with conventional iPSC generation methods that rely on ectopic overexpression of pluripotency transcription factors (e.g., Oct4, Sox2, Klf4, c-Myc). These conventional approaches involve global chromatin remodeling with poor precision, unclear necessity of simultaneous remodeling of multiple loci, and potential safety concerns from exogenous factor expression. It is unknown whether precise remodeling of single endogenous loci is sufficient to induce pluripotency and how distal enhancer elements contribute. This invention provides a more precise, efficient, and potentially safer reprogramming strategy by using a CRISPR-based activation system to modify endogenous loci.
The disclosure demonstrates that targeting and remodeling selective endogenous gene loci, including single loci such as Sox2 promoter alone or both Oct4 promoter and enhancer simultaneously, using sgRNAs and a CRISPRa system, is sufficient to initiate cellular reprogramming of somatic cells to authentic iPSCs with pluripotent gene expression, morphology, and differentiation potential. The CRISPRa system can utilize a dCas9 fused to VP64 or to a SunTag array recruiting multiple transcriptional activators, and/or the acetyltransferase domain of p300 (p300core) to modify histone acetylation. This epigenetic remodeling at specific loci efficiently triggers upregulation of pluripotency genes and establishment of pluripotency networks without ectopic factor overexpression.
Claims Coverage
The patent includes multiple independent claims covering methods of generating iPSC populations by targeting endogenous gene loci with CRISPR activation systems, focusing on specific sgRNAs targeting promoters and enhancers of pluripotency genes and the structure of the CRISPRa system used.
Method of generating iPSCs by targeting Oct4 promoter and enhancer with multiple sgRNAs
A method that uses at least two sgRNAs targeting an Oct4 promoter and an Oct4 enhancer in non-iPSC cells, employing a CRISPR activation system to remodel the loci and increase the number of generated iPSCs compared to without targeting the enhancer.
Extension of targeted gene loci to include multiple pluripotency-associated genes
The method further targets additional endogenous loci such as Sox2, Klf4, c-Myc, Nanog, Lin28, Nr5a2, Glis1, Cebpa, or combinations thereof, using sgRNAs specific for their promoter or regulatory regions to enhance iPSC generation.
Use of specific sgRNA sequences for targeting gene promoters and enhancers
Employing sgRNAs with nucleotide sequences corresponding to identified SEQ ID NOs for target genes' promoter or enhancer regions, ensuring specificity and efficacy of gene activation.
CRISPR activation system comprising deactivated Cas fused to transcriptional activators
Utilization of a CRISPR activation system that includes a nuclease-deactivated Cas protein fused to transcriptional activators, optionally employing a tandem peptide array such as SunTag to enhance activator recruitment.
Specific structural components of the CRISPR activation system
The CRISPR activation system comprises dCas9, a SunTag tandem peptide array fused to dCas9, and transcriptional activators such as herpes simplex VP16, VP64, or p65 domains.
Cell types applicable for reprogramming using the method
The method is applicable to populations of non-iPSC cells including fibroblasts, skin cells, cord blood cells, peripheral blood cells, renal epithelial cells, mammalian cells, and human cells.
Inclusion of small molecules to enhance reprogramming efficiency
The method optionally includes contacting non-iPSC cells with small molecule inhibitors (TGFβR inhibitor, GSK3 inhibitor, MEK inhibitor, ROCK inhibitor) and further culturing generated iPSCs with selected inhibitors excluding TGFβR inhibitor to improve reprogramming.
Specific CRISPR activation system employing dCas9-SunTag and p300core for epigenetic remodeling
A method using CRISPR activation system comprised of dCas9 fused to a SunTag array and attached to the acetyltransferase activity domain of p300 (p300core) to remodel histone acetylation at endogenous gene loci (e.g., Oct4 promoter and enhancer) to trigger iPSC generation.
The claims collectively cover novel methods for generating iPSCs by precise targeting and remodeling of endogenous pluripotency gene loci using a CRISPR activation system with defined sgRNAs, with an emphasis on combinatorial promoter and enhancer targeting and structural features of the CRISPRa system to increase reprogramming efficiency in various somatic cell types.
Stated Advantages
Provides a precise and efficient method to reprogram somatic cells into iPSCs without reliance on ectopic expression of transcription factors.
Enables reprogramming by targeting single endogenous gene loci or combinations, reducing complexity and potential off-target effects.
Utilizes epigenetic remodeling of endogenous gene promoters and enhancers, offering a safer and more controlled approach.
Allows increased reprogramming efficiency and decreased variability, especially when using secondary MEFs derived from CRISPRiPSCs.
Compatible with small molecule treatments to further enhance reprogramming efficiency.
Documented Applications
Reprogramming of adult somatic cells including fibroblasts, skin cells, cord blood cells, peripheral blood cells, and renal epithelial cells into iPSCs.
Use in both mouse and human cell reprogramming contexts, as demonstrated with MEFs, tail tip fibroblasts, and human dermal fibroblasts.
Generation of pluripotent stem cells for use in regenerative medicine, basic biological research, and disease modeling.
Potential adaptation for reprogramming other cell types and application in various model systems.
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