Inference of CRISPR Edits from Sanger Trace Data

Conant, David S.; Hsiau, Tim; Rossi, Nicholas A. A.; Oki, Jennifer; Maures, Travis J.; Waite, Kelsey; Yang, Jason; Joshi, Sahil; Kelso, Reed; Holden, Kevin; Enzmann, Brittany L.; Stoner, Rich

doi:10.1089/crispr.2021.0113

Cited by 357 publications

(299 citation statements)

References 19 publications

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“…The efficient introduction of nonsense mutations within edited cells was confirmed by Sanger sequencing and waveform decomposition analysis ( Supplementary Fig. S1A-B ) (Conant et al 2022). We estimate that ∼60% of alleles were successfully edited ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 93%

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

Moll

Odon

Harvey

et al. 2022

Preprint

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New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase (OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

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Section: Resultsmentioning

confidence: 93%

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

Moll

Odon

Harvey

et al. 2022

Preprint

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“…To evaluate if risks of off-target effects increased with the routine usage of two or three different gRNA target sequences in iCAP genome editing method, we performed targeted amplicon sequencing of 50 off-target sites which present 4 or 5 the most closely mismatched off-target sequences for each of 11 different gRNA target sequences used for the study. We used ICE (Inference of C RISPR E dits) algorithm tool (Conant, et al, 2022) to analyze the sequencing data for evidence of indel mutagenesis at the sites examined. The assessment reports show indel rates of zero percent at these top off-target sites suggesting no off-target effects detected and R 2 values of 0.95-1.0 indicating “goodness of fit” of the analysis quality (Table S3).…”

Section: Resultsmentioning

confidence: 99%

“…PCR amplicons produced from experimental (edited) and control (unedited or wildtype) genomic DNA samples were Sanger Sequenced by GeneWiz sequencing service. DNA sequences were then compared & analyzed by using the online I nference of C RISPR E dits (ICE) algorithm tool (Conant, et al, 2022) (https://www.synthego.com/products/bioinformatics/crispr-analysis). A report of ICE analysis provides an R 2 value and indel rate.…”

Section: Methodsmentioning

confidence: 99%

An in situ cut-and-paste genome editing platform mediated by CRISPR/Cas9 or Cas12a

Jiang

Kemper

Chang

et al. 2022

Preprint

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Recombinant DNA technology mediated by restriction enzymes and ligases allows in vitro manipulation of a DNA segment isolated from the genome. Short overhangs generated by restriction enzymes facilitate efficient pasting together a DNA sequence and a vector. We adopted this recombinant DNA strategy to develop an in vivo recombinant-genome genome editing approach. Using the programmable endonuclease Cas9 or Cas12a as a restriction enzyme, we devised an in situ cut-and-paste (iCAP) genome editing method that was tested in both mouse germline and human cell line platforms. Mouse gene loci Slc35f2 and Slc35f6 were each edited with in-frame insertion of a large APEX2-Cre cassette and concurrent FRT3 insertion at a second location providing proof of principle for the iCAP method. Further, a de nova single nucleotide mutation associated with MED13L syndrome was efficiently corrected in patient cells. Altogether, the iCAP method provides a single genome editing platform with flexibility and multiutility enabling versatile and precise sequence alterations, such as insertion, substitution, and deletion, at single or multiple locations within a genomic segment in mammalian genomes.

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“…The same amounts of gRNA were used in all experimental groups. The efficiency of gRNA was checked by analyzing the sequence of the target sequence with the ICE CRISPR Analysis Tool (Conant et al, 2022) after amplification using a specific pair of primers (Table S2).…”

Section: Silica Crystal Injection and Gene Editionmentioning

confidence: 99%

Combination therapy targeting inflammasome and fibrogenesis alleviates inflammation and fibrosis in a zebrafish model of silicosis

Tyrkalska

Pedoto

Martínez‐López

et al. 2022

Preprint

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Silicosis is a long-term lung disease caused by the inhalation of large amounts of crystalline silica dust. As there is no effective treatment available, patients are provided with supportive care, and some may be considered for lung transplantation. There is therefore an evident need for a better understanding of the disease’s biology and for identifying new therapeutic targets and therapies. In this context, our group has developed a larval zebrafish model of silicosis by injecting silica crystals into the hindbrain ventricle, a cavity into which immune cells can be recruited and that mimics the alveolar environment of the human lung. The injection of silica crystals into this cavity led to the initiation of local and systemic immune responses driven through both TLR- and inflammasome-dependent signaling pathways, followed by fibrosis, as happens in human patients. The combination of the inflammasome inhibitor VX-765 and the antifibrotic agent pirfenidone was found to be the best therapy to alleviate both inflammation and fibrosis. The zebrafish model of silicosis developed here is a unique tool that will shed light onto the molecular mechanisms involved in the progression of this devastating disease and for identifying novel drugs that improve the quality of life of silicosis patients.

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Inference of CRISPR Edits from Sanger Trace Data

Cited by 357 publications

References 19 publications

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication

An in situ cut-and-paste genome editing platform mediated by CRISPR/Cas9 or Cas12a

Combination therapy targeting inflammasome and fibrogenesis alleviates inflammation and fibrosis in a zebrafish model of silicosis

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