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Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

ABSTRACT: Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

PROVIDER: PRJNA1011332 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25841400.fastq.gz	Fastqsanger.gz
	SRR25841401.fastq.gz	Fastqsanger.gz
	SRR25848706.fastq.gz	Fastqsanger.gz
	SRR25848707.fastq.gz	Fastqsanger.gz
	SRR25848708.fastq.gz	Fastqsanger.gz

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Project description:Alternative splicing is an essential mechanism for diversifying proteins, with different mature RNA isoforms producing different proteins with potentially distinct functions. Two major challenges in characterizing the cellular function of isoforms are the lack of both experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design. To address these gaps, we developed and methodically tested a strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs (gRNAs) that overlap exon-exon junctions in the mature RNA. We perform a high-throughput essentiality screen, numerous qPCR assays, and PacBio long read sequencing to affirm that our strategy works to specifically target the unique junctions of isoforms to robustly knockdown their RNA. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions in current GENCODE annotations and our predictions of gRNA efficacy, we estimate that this strategy can target 94% of all human isoforms, including 29,238 protein-coding and 9,638 lncRNA isoforms that are specifically targetable with a gRNA spanning a unique splice junction.

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Project description:Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we developed and methodically tested an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. Using the example gene RBFOX2, we validated protein-level changes and assessed the functional impact of isoform-specific knockdown. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all transcriptional and splicing events, including exon skipping and inclusion, alternative 5’ and 3’ splice sites, and alternative starts and ends.

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