Computational design of sequence-specific DNA-binding proteins
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ABSTRACT:
PROVIDER: PRJNA993300 | ENA |
REPOSITORIES: ENA
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Project description:Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove.
2024-07-05 | GSE237017 | GEO
Project description:CLIP1 and DMD are two novel RNA-binding proteins through computational prediction and experimental validation
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Project description:RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific
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Project description:Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)—proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.
2023-03-31 | GSE215198 | GEO
Project description:Since RBPs play important roles in the cell, it’s particularly important to find new RBPs. We performed iRIP-seq and CLIP-seq to verify two proteins, CLIP1 and DMD, predicted by RBPPred whether are RBPs or not. The experimental results confirm that these two proteins have RNA-binding activity. We identified significantly enriched binding motifs UGGGGAGG, CUUCCG and CCCGU for CLIP1 (iRIP-seq), DMD (iRIP-seq) and DMD (CLIP-seq), respectively. The computational KEGG and GO analysis show that the CLIP1 and DMD share some biological processes and functions. Besides, we found that the SNPs between DMD and its RNA partners may associate with Becker muscular dystrophy, Duchenne muscular dystrophy, Dilated cardiomyopathy 3B and Cardiovascular phenotype. Among the thirteen cancers data, CLIP1 and another 300 genes always co-occur, and 123 of these 300 genes interact with CLIP1. These cancers may be associated with the mutations in both CLIP1 and the genes it interacts with.
2021-12-15 | GSE128318 | GEO
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.
2023-09-13 | GSE242106 | GEO
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.
2023-09-13 | GSE242105 | GEO
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.
2023-09-13 | GSE242104 | GEO