Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Tsai, Yu‐Chih; Greenberg, David; Powell, James; Höijer, Ida; Ameur, Adam; Strahl, Maya; Ellis, Ethan; Jonasson, Inger; Pinto, Ricardo Mouro; Wheeler, Vanessa C.; Smith, Melissa; Gyllensten, Ulf; Sebra, Robert; Korlach, Jonas; Clark, Tyson A.

doi:10.1101/203919

Cited by 51 publications

(60 citation statements)

References 43 publications

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“…Our methods are inspired by previously proposed assays where a single gRNA was used to perform targeted enrichment. SMRT-OTS is a modified version of a protocol we previously applied for detection of repeat expansions in human cell lines and blood samples 30,31 , while Nano-OTS is adapted from a targeted sequencing assay 32 used for detection of unknown fusion gene partners 33 . In addition to wet lab assays, we developed a computational method that can be used to identify gRNA binding sites a single base pair resolution, both from high-quality SMRT reads and from lower quality nanopore reads (see Figure 1C).…”

Section: Two New Amplification-free Protocols For Off-target Sequencingmentioning

confidence: 99%

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Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Höijer

Johansson

Gudmundsson

et al. 2020

Preprint

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A much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on-and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 highconfidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplificationfree sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

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Section: Two New Amplification-free Protocols For Off-target Sequencingmentioning

confidence: 99%

“…SMRT-OTS libraries were prepared in a similar manner described by Tsai et al 30 Asymmetric SMRTbell template sequencing data was subjected to a customized analysis pipeline for capture and conventional hairpin-adapter recognition for separating subreads.…”

Section: Smrt-ots: Off-target Sequencing Using Pacbio's Smrt Sequencingmentioning

confidence: 99%

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Höijer

Johansson

Gudmundsson

et al. 2020

Preprint

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“…Among those aforementioned engineered DNA-binding molecules, the CRISPR/Cas9 system is the most convenient, economical and time-efficient, and has rapidly risen to prevail over technologies with similar functions 9,10 . CRISPR/Cas9 has been used recently for targeted sequencing microsatellite-spanning sequences 11 and loci associated with repeat expansion disorders in conjunction with Pacific Biosystems’ Single Molecule Real Time (SMRT) sequencing 12 .…”

Section: Introductionmentioning

confidence: 99%

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

Slesarev

Viswanathan

Tang

et al. 2018

Preprint

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The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines, and compare it to popular target-enrichment methods and to whole genome sequencing (WGS). The CRISPR/Cas9-based techniques allow for amplification-free capture of genomic regions, which reduces the possibility of sequencing artifacts. Other advantages of these methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of longer sequencing templates for real-time sequencing. The utility of these protocols has been proven by identification of transgene integration sites and flanking sequences in a number of CHO cell lines. However, data produced by these and other targeted capture methods are not always sufficient to analyze complex genomic rearrangements (CGRs) or unexpected sequences introduced into genome by vector integration events. In contrast, WGS provides complete information about vector integration sites, vector copy number, CGRs, and foreign DNA-but despite these benefits, WGS is not easily implemented due to the cost and complexity of the analysis.

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“…Here, we demonstrate that both PacBio and ONT sequencing platforms can sequence through repeats cloned into plasmids, including the spinocerebellar ataxia type 36 (SCA36) disease-causing ‘ GGCCTG’ repeat expansion [20] and the FTD- and ALS-causing G 4 C 2 repeat expansion. We further report long-read sequencing data from the C9orf72 G 4 C 2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using both whole-genome and no-amplification (No-Amp) targeted sequencing [21,22] on the PacBio Sequel. Our findings indicate that long-read sequencing is well suited to characterizing repeat expansions and that this technology has potential to accelerate future genetic discovery efforts across a broad range of diseases that may involve repeat expansions.…”

Section: Introductionmentioning

confidence: 99%

Long-read sequencing across theC9orf72‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

Ebbert

Farrugia

Sens

et al. 2018

Preprint

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Background: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinlON was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8x coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >800x coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >99% G4C2 content, though we cannot rule out small interruptions.Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

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Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Cited by 51 publications

References 43 publications

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

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

Long-read sequencing across theC9orf72‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

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