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.
2013-04-09 | E-GEOD-45770 | biostudies-arrayexpress
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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Project description:Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor nerve cells, muscle weakness and atrophy. While current treatment strategies including small molecules or viral vectors improved motor function and survival, an ultimate treatment is highly challenging to correct the endogenous mutations by genome-editing technologies. We have developed the CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, which allowed for DNA knock-in in both dividing and non-dividing cells in vivo. Here, we demonstrated a gene correction of mutant mSmn1 by using the HITI strategy to treat SMA model mice. To provide the long-term therapeutic benefits of gene correction, we established a unique approach to treat SMA by combination with gene cDNA supplementation and editing (Gene-DUET). As a proof of concept of therapeutic approach for SMA, we demonstrated the advantageous effects including motor function and survival improvement by Gene-DUET strategy in SMA model mice. The Gene-DUET represents a powerful strategy for treating inherited diseases especially for neurodegenerative disorders as well as neuromuscular diseases.
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