Crispr/Cas9 Targeted Capture of Mammalian Genomic Regions for Characterization by NGS

Slesarev, Alexei I.; Viswanathan, Lakshmi; Tang, Yitao; Borgschulte, Trissa; Achtien, Katherine; Razafsky, David; Onions, David; Chang, Audrey; Côté, Colette

doi:10.1101/372987

Cited by 4 publications

(7 citation statements)

References 46 publications

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“…This has motivated recent efforts to enrich loci of interest for subsequent LRS without amplification, which limits target-lengths and can introduce PCR bias. Many emerging methods leverage the highly specific targeting ability of the CRISPR/Cas9 system, but strategies vary widely and have unique strengths and limitations related to DNA input requirements, protocol execution time, target size restrictions, and efficiency (16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

Wallace

Sasani

Swanier

et al. 2020

Preprint

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A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a novel method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in up to 416X coverage of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

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Section: Introductionmentioning

confidence: 99%

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

Wallace

Sasani

Swanier

et al. 2020

Preprint

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“…Our method is the latest in an expanding suite of CRISPR-based targeted sequencing approaches. Some methods seek to characterize repeat expansions (4, 13) or fusion genes (5, 6), while others are aimed at enriching one or a few loci (8, 11, 14, 15). The published approaches that are most comparable to ours are CRISPR-Cap (10) and nCATS (7).…”

Section: Discussionmentioning

confidence: 99%

Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

Mighell

Nishida

et al. 2020

Preprint

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Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

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“…This signature is similar to those observed with DIGENOME-Seq 23 , SITE-Seq 28 , and DISCOVER-Seq 22 , which allowed us to use previously developed software for the analysis of RGEN-seq experimental data 22,25,28 . RGEN-Seq was extensively tested on a Chinese hamster CHO-K1 clone bearing a 180 kb E. coli insert 30 . Isolated genomic DNA was targeted with seven sgRNAs, six of which tile across a 40 kb portion of the integrated E. coli region (Fig.…”

Section: Rgen-seq Methodsmentioning

confidence: 99%

Rgen-Seq for Highly Sensitive Amplification-Free Screen of Off-Target Sites of Gene Editors

Kuzin

Redler

Onuska

et al. 2021

Preprint

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Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, they still leave plenty of room for improvement. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. The method is extremely simple, and it is on a par or even supersedes in sensitivity existing assays without reliance on amplification steps. The latter saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

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Crispr/Cas9 Targeted Capture of Mammalian Genomic Regions for Characterization by NGS

Cited by 4 publications

References 46 publications

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

Rgen-Seq for Highly Sensitive Amplification-Free Screen of Off-Target Sites of Gene Editors

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