CRISPAltRations: A validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing

Kurgan, Gavin; Turk, Rolf; Li, Heng; Roberts, Nathan James; Rettig, Garrett R.; Jacobi, Ashley M.; Tso, Lauren; Sturgeon, Morgan; Mertens, Massimo; Noten, Roel; Florus, Kurt; Behlke, Mark A.; Wang, Yu; McNeill, Matthew

doi:10.1016/j.omtm.2021.03.024

Cited by 20 publications

(12 citation statements)

References 66 publications

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“…Illumina datasets feature an average quality score higher than Nanopore, thus a few hundred successfully aligned reads should be sufficient for genotyping purposes ( 13 ). By contrast, sequencing bulk of cells will require a higher number of reads ( 14 ), in our setup ∼2000 reads, and the sequencing coverage level will determine whether low frequency indel discovery can be made with a certain degree of confidence.…”

Section: Resultsmentioning

confidence: 99%

Identification of genome edited cells using CRISPRnano

Nguyen

Ramachandran

Martins

et al. 2022

Nucleic Acids Research

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Genome engineering-induced cleavage sites can be resolved by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Identifying genetically modified clones at the target locus remains an intensive and laborious task. Different workflows and software that rely on deep sequencing data have been developed to detect and quantify targeted mutagenesis. Nevertheless, these pipelines require high-quality reads generated by Next Generation Sequencing (NGS) platforms. Here, we have developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (www.CRISPRnano.de) that enables the analysis of low-quality reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with NGS and ONT sequencing reads, and it can be used without an internet connection.

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Section: Resultsmentioning

confidence: 99%

Identification of genome edited cells using CRISPRnano

Nguyen

Ramachandran

Martins

et al. 2022

Nucleic Acids Research

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“…In recent years, several tools have been created to analyse amplicon data, such as AmpliCan ( 7 ), CRISPResso2 ( 6 ), CrispRVariants ( 29 ), ScarMapper ( 19 ) and CRISPAltRations ( 30 ). We found SIQ to perform on par with CRISPResso2 and AmpliCan, and both to perform slightly better than ScarMapper on Cas9-induced breaks.…”

Section: Discussionmentioning

confidence: 99%

SIQ: easy quantitative measurement of mutation profiles in sequencing data

Schendel

Schimmel

Tijsterman

2022

NAR Genomics and Bioinformatics

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With the emergence of CRISPR-mediated genome editing, there is an increasing desire for easy-to-use tools that can process and overview the spectra of outcomes. Here, we present Sequence Interrogation and Quantification (SIQ), a simple-to-use software tool that enables researchers to retrieve, data-mine and visualize complex sets of targeted sequencing data. SIQ can analyse Sanger sequences but specifically benefit the processing of short- and long-read next-generation sequencing data (e.g. Illumina and PacBio). SIQ facilitates their interpretation by establishing mutational profiles, with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using insightful tornado plot visualizations as outputs, we illustrate that SIQ readily identifies sequence- and repair pathway-specific mutational signatures in a variety of model systems, such as nematodes, plants and mammalian cell culture.

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“…Each sample was barcoded and sequenced using the Illumina sequencing platform. Demultiplexed NGS data was analyzed for genome editing with CRISPAltRations v1.0.0 using default parameters and the recommended window size (9 bp) for detecting Cas12a editing ( Kurgan et al, 2020 ). All primers used for RFLP assay and amplicon deep sequencing are listed in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases

et al. 2022

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

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CRISPAltRations: A validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing

Cited by 20 publications

References 66 publications

Identification of genome edited cells using CRISPRnano

Identification of genome edited cells using CRISPRnano

SIQ: easy quantitative measurement of mutation profiles in sequencing data

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases

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