Optimizing genome editing strategy by primer-extension-mediated sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA477827 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7421563_1.fastq.gz | Fastqsanger.gz | |||
| SRR7421563_2.fastq.gz | Fastqsanger.gz | |||
| SRR7421564_1.fastq.gz | Fastqsanger.gz | |||
| SRR7421564_2.fastq.gz | Fastqsanger.gz | |||
| SRR7421565_1.fastq.gz | Fastqsanger.gz |
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Project description:High specificity of e\ngineered nucleases ensures precise genome editing. Couple methods were developed to identify off-target sites of CRISPR/Cas9, but hardly any high-throughput sequencing method can unequivocally determine their targeting efficiencies. Here we describe a comprehensive method, primer-extension-mediated sequencing (PEM-seq), which could sensitively detect CRISPR/Cas9 off-target sites as well as assess their editing efficiency by quantifying DNA interference events at on-target sites. Demonstrated by PEM-seq, we generated a high-fidelity Cas9 variant FeCas9 that possesses similar targeting ability as the wild-type while with extremely low off-target activities. Moreover, we provided further evidences for the broader range of xCas9 protospacer adjacent sequence. We also found the AcrIIA4 inhibitor could inhibit both on- and off-target activities of SpCas9, but it suppressed SpCas9 cleavage at the off-target loci not so efficiently as at the on-target sites. Finally, we believe PEM-seq is applicable to optimizing genome editing strategy for clinical purpose or creating animal model.
2019-04-03 | GSE116231 | GEO
Project description:Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
| S-EPMC6434046 | biostudies-literature
Project description:New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2â²-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems. We sequenced in-vitro transcribed RNaseP in wild type and barcoded variants and probed them with SHAPE-Seq. Comparison to existing crystalography data and previous SHAPE experiments verified the accuracy of the technique and the absence of bias due to our multiplexiing strategy.
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Project description:Nonsyndromic cleft lip with or without palate (NSCL/P) are common birth defects of complex etiology. Multiple interacting loci with possible additional environment factors influence the risk for NSCL/P. 12 single nucleotide polymorphisms (SNPs) in 7 candidate genes were tested using an allele-specific primer extension for both case-control and case-parents analyses in northeast China (236 unrelated patients, 185 mothers and 154 fathers, including 128 complete trios, 400 control individuals). TGFA and IRF6 genes showed a significant associations with NSCL/P. For IRF6, statistical evidence of association between rs2235371 (P=0.003), rs2013162 (P=0.000) and NSCL/P was found in case-control analyses. The Family Based Association Tests (FBAT) showed an over-transmission for C allele of rs2235371 polymorphic (p=0.007). For TGFA, associations between rs3771494, rs3771523 (G3822A), rs11466285 (T3851C) and NSCL/P could be observed both in case-control and FBAT analyses. However, we found no associations between other genes (BCL3, TGFB3, MTHFR1, PVRL1 and SUMO1) and NSCL/P. In the study present, 236 unrelated patient with NSCL/P addition with 185 mothers and 154 fathers and 400 control individuals were tested with the allele special primer extension microarray.
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Project description:New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2â²-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.
2011-08-26 | GSE31573 | GEO
Project description:Optimizing CRISPR/Cas9 editing of repetitive single nucleotide variants
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Project description:Nonsyndromic cleft lip with or without palate (NSCL/P) are common birth defects of complex etiology. Multiple interacting loci with possible additional environment factors influence the risk for NSCL/P. 12 single nucleotide polymorphisms (SNPs) in 7 candidate genes were tested using an allele-specific primer extension for both case-control and case-parents analyses in northeast China (236 unrelated patients, 185 mothers and 154 fathers, including 128 complete trios, 400 control individuals). TGFA and IRF6 genes showed a significant associations with NSCL/P. For IRF6, statistical evidence of association between rs2235371 (P=0.003), rs2013162 (P=0.000) and NSCL/P was found in case-control analyses. The Family Based Association Tests (FBAT) showed an over-transmission for C allele of rs2235371 polymorphic (p=0.007). For TGFA, associations between rs3771494, rs3771523 (G3822A), rs11466285 (T3851C) and NSCL/P could be observed both in case-control and FBAT analyses. However, we found no associations between other genes (BCL3, TGFB3, MTHFR1, PVRL1 and SUMO1) and NSCL/P.
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