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

Höijer, Ida; Johansson, Josefin; Gudmundsson, Sanna; Chin, Chen-Shan; Bunikis, Ignas; Häggqvist, Susana; Emmanouilidou, Anastasia; Wilbe, Maria; Hoed, Marcel den; Bondeson, Marie Louise; Feuk, Lars; Gyllensten, Ulf; Ameur, Adam

doi:10.1101/2020.02.09.940486

Cited by 17 publications

(25 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subsequently, the CCS tool in SMRT analysis was used to create circular consensus sequencing reads from the subreads. A detailed step-by-step instruction of the SMRT-OTS protocol is available on protocols.io ( https://www.protocols.io/view/smrt-ots-bjugkntw ) [ 52 ].…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

Background One ongoing 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 activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Sequencing was performed using the MinION system (Oxford Nanopore Technologies) with a R9.4.1 flow cell and Guppy v3.3.3 was used for base calling. A detailed step-by-step instruction of the Nano-OTS protocol is available on protocols.io ( https://www.protocols.io/view/nano-ots-bjmukk6w ) [ 53 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Nanopore off-target sequencing. We next used in vitro Nanopore off-target sequencing (Nano-OTS) 26 in DNA from an adult Tg(acta2:GFP) positive fish used to generate CRISPR/Cas9 founders to explore if offtarget mutagenic activity may have influenced the results for hcn4; and if it may have played a role in the relatively large number of missed sequencing calls for neo1b (Supplementary Table S4). No off-target mutations were identified for the hcn4 and neo1b gRNAs.…”

Section: Effect Of Mutations In Candidate Genes On Body Sizementioning

confidence: 99%

“…Genomic DNA was fragmented to 20 kb using Megaruptor 2 (Diagenode, Liege, Belgium) and size selected with a 10 kb cut-off using the Blue Pippin system (Sage Science, Beverly, MA). Libraries for Nano-OTS were prepared as described recently by Höijer et al 26 . In short, ribonucleoproteins (RNPs) were prepared as a pool using the hcn4, hcn4l, neo1a and neo1b gRNAs.…”

Section: Quality Control Of Phenotype Datamentioning

confidence: 99%

See 1 more Smart Citation

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach

Heyde

Emmanouilidou

Mazzaferro

et al. 2020

Sci Rep

Self Cite

View full text Add to dashboard Cite

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image-and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine-an open channel blocker of HCNs-for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755). Heart rate variability (HRV) reflects the inter-beat variation of the RR interval. HRV is controlled by the sinoatrial node, which receives input from the autonomic nervous system. Autonomic imbalance has been associated with work stress and other modifiable or non-modifiable risk factors 1 , and is reflected in lower HRV. Lower HRV has been associated with higher cardiac morbidity and mortality 2 , as well as with a higher risk of all-cause mortality 3. HRV can be quantified non-invasively using an ECG, making HRV a useful clinical marker for perturbations of the autonomic nervous system. However, the mechanisms influencing HRV remain poorly understood. Recently, we and others identified the first loci that are robustly associated with HRV, using a meta-analysis of genome-wide association studies (GWAS) with data from 53,174 participants 3. Five of the identified loci had

show abstract

Determining On‐Target, Off‐Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe‐Harbor Modification of iPSCs Using Double‐Control Quantitative Copy Number PCR

Schjeide

Püschel

2023

Current Protocols

View full text Add to dashboard Cite

Double‐control quantitative copy number PCR (dc‐qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert‐confirmation PCR, the technique can quickly and easily identify clones containing on‐target heterozygous or homozygous donor DNA integrations and exclude off‐target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription‐activator‐like effector nucleases (TALENs), and zinc‐finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost‐prohibitive or create dependency on manufacturers for assay design and production. The dc‐qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step‐by‐step instructions to prepare and perform the dc‐qcnPCR, and its companion insert‐confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90‐4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert‐confirmation PCR Basic Protocol 3: Design and preparation of double‐control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells

show abstract

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

Cited by 17 publications

References 47 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

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach

Determining On‐Target, Off‐Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe‐Harbor Modification of iPSCs Using Double‐Control Quantitative Copy Number PCR

Contact Info

Product

Resources

About