DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks

Overbeek, Megan van; Capurso, Daniel; Carter, Matthew M.; Thompson, Matthew S.; Frias, Elizabeth; Russ, Carsten; Reece‐Hoyes, John S.; Nye, Christopher H.; Gradia, Scott; Vidal, Bastien; Zheng, Jiashun; Hoffman, Gregory R.; Fuller, Christopher K.; May, Andrew P.

doi:10.1016/j.molcel.2016.06.037

Cited by 419 publications

(440 citation statements)

References 42 publications

(47 reference statements)

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“…http://dx.doi.org/10.1101/392217 doi: bioRxiv preprint first posted online Aug. 15, 2018; Analysis of Cas9-treated samples at each guide target location suggests that the position of the most-frequently deleted bases varied between genomic loci, a finding which is similar to previous descriptions of guide-specific deletion profiles 19 . The HEK2, EMX1, and FANCF guides induced deletions at position 17 (from the 5' end of the guide target), while Cas9-mediated deletion activity is predominantly observed at position 16 for the HEK3 and HEK4 guides (Supplementary Fig.…”

supporting

confidence: 78%

Analysis and comparison of genome editing using CRISPResso2

Clement

Rees

Canver

et al. 2018

Preprint

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Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or offtarget regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/392217 doi: bioRxiv preprint first posted online Aug. 15, 2018; 2 The field of genome editing is rapidly advancing, and the technologies to modify the genome are becoming increasingly more accurate, efficient and versatile 1 . For example, base editors-a recent class of genome editing technology-harness the targeting properties of RNA-guided endonucleases to precisely change one nucleotide in a predictable manner 2,3,4 . As sequencing costs decrease and access to next-generation sequencing machines becomes more widespread, targeted amplicon sequencing is becoming the gold standard for the validation and characterization of genome editing experiments.CRISPResso2 introduces five key innovations for the analysis of genome editing data: (1) Comprehensive analysis of sequencing data from base editors; (2) Allele specific quantification of heterozygous references; (3) A novel biologically-informed alignment algorithm; (4) Ultra-fast processing time; and (5) A batch mode for analyzing and comparing multiple editing experiments.Existing software packages for the analysis of data generated by genome editing experiments are designed to only analyze cleavage events resulting from nuclease activity 5,6,7,8,9,10 . CRISPResso2 (http://crispresso2.pinellolab.org) is the first comprehensive software specifically designed to analyze base editor data from amplicon sequencing, in addition to quantifying and visualizing indels from other nucleases. CRISPResso2 allows users to readily quantify and visualize amplicon sequencing data from base editing experiments. It takes in raw FASTQ sequencing files as input and outputs reports describing frequencies and efficiencies of base editing activity, plots showing base substitutions across the entire amplicon region (Fig. 1a) and nucleotide substitution frequencies for a region specified by the user (Fig. 1b). Additionally, users can specify the nucleotide substitution (e.g., C->T or A->G) that is relevant for the base editor used, and publication-quality plots are produced for nucleotides of interest with a heatmap showing conversion efficiency (Fig. 1c). Reads (left) can be assigned to each allele using CRISPResso2 (right) to achieve accurate quantification of genome editing at genomic loci with mu...

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supporting

confidence: 78%

Analysis and comparison of genome editing using CRISPResso2

Clement

Rees

Canver

et al. 2018

Preprint

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“…However, endonuclease activity can also have undesired on-target effects. Notably, nonhomologous end joining (NHEJ) downstream of Cas9-mediated cleavage is associated with an increased risk of indel formation (van Overbeek et al 2016). This has prompted the development of Cas9 derivatives that nick rather than cleave DNA (Cong et al 2013; Mali et al 2013), shifting repair pathway choice away from NHEJ and toward HR.…”

mentioning

confidence: 99%

Mobile Introns Shape the Genetic Diversity of Their Host Genes

Repar

Warnecke

2017

Genetics

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“…This was important since CRISPR/Cas9 leads to various indel sizes [19], thus some might be more or less easily discriminated (due to differences in inner primer affinity), and we thought that optimization was required to enable discrimination of all of them from wild type. We first tested whether there is an optimal orientation of the inner primers (Fig 2A).…”

Section: Resultsmentioning

confidence: 99%

Detection of genome-edited mutant clones by a simple competition-based PCR method

Harayama

Riezman

2017

PLoS ONE

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Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.

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DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks

Cited by 419 publications

References 42 publications

Analysis and comparison of genome editing using CRISPResso2

Analysis and comparison of genome editing using CRISPResso2

Mobile Introns Shape the Genetic Diversity of Their Host Genes

Detection of genome-edited mutant clones by a simple competition-based PCR method

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