Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA602699 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10952000.fastq.gz | Fastqsanger.gz | |||
| SRR10952001.fastq.gz | Fastqsanger.gz | |||
| SRR10952002.fastq.gz | Fastqsanger.gz | |||
| SRR10952003.fastq.gz | Fastqsanger.gz | |||
| SRR10952004.fastq.gz | Fastqsanger.gz |
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Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
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Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
2020-02-29 | GSE144078 | GEO
Project description:Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform
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Project description:The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypic Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated for the first time the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
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Project description:Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with strong regulatory effects on gene expression. Analysis of NRF1 alternative exon-7 revealed that this exon modulates NRF1 transcriptional activity by regulating its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.
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Project description:Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with strong regulatory effects on gene expression. Analysis of NRF1 alternative exon-7 revealed that this exon modulates NRF1 transcriptional activity by regulating its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.
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Project description:Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with strong regulatory effects on gene expression. Analysis of NRF1 alternative exon-7 revealed that this exon modulates NRF1 transcriptional activity by regulating its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.
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Project description:Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with strong regulatory effects on gene expression. Analysis of NRF1 alternative exon-7 revealed that this exon modulates NRF1 transcriptional activity by regulating its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.
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Project description:Cas12a CRISPR technology, unlike Cas9, allows for multiplexing guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.
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