Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Bae, Sangsu; Park, Jeongbin; Kim, Jin‐Soo

doi:10.1093/bioinformatics/btu048

Cited by 1,805 publications

(1,430 citation statements)

References 17 publications

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“…The potential off‐target sites were predicted using the online tool Cas‐OFFinder (Bae et al ., 2014). The sites of each target in five randomly selected mutant lines were examined by site‐specific PCR and direct Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Qin

et al. 2017

Plant Biotechnology Journal

231

152

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Summary CRISPR‐Cpf1 is a newly identified CRISPR‐Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR‐Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR‐Cpf1. We found that pre‐crRNAs with a full‐length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA‐Cpf1‐induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR‐Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.

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Section: Methodsmentioning

confidence: 99%

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Qin

et al. 2017

Plant Biotechnology Journal

231

152

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“… a , Number of in silico predicted target sites mismatched by ‘n’ positions for six sgRNAs against the reference human genome (hg38) via Cas-OFFinder 29 . b , Assessment of GUIDE-seq dsODN tag integration at the on-target site for each nuclease and guide combination, detected by RFLP assay.…”

Section: Extended Datamentioning

confidence: 99%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

971

877

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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“…PCR amplicons were subjected to paired-end sequencing using Illumina Miniseq. Cas-analyzer was used for analysing indel and HDR frequencies 46,47 . The primers used for amplification are listed in Extended Data Table 5.…”

Section: Article Researchmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

819

540

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More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

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Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Cited by 1,805 publications

References 17 publications

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Correction of a pathogenic gene mutation in human embryos

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