Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq

2021 ◽  
Author(s):  
Nikolay L. Malinin ◽  
GaHyun Lee ◽  
Cicera R. Lazzarotto ◽  
Yichao Li ◽  
Zongli Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Nuclease Activity ◽  
Genome Wide

scholarly journals NickSeq for genome-wide strand-specific identification of DNA single-strand break sites with single nucleotide resolution

2019 ◽  
Author(s):  
Joshua J. Elacqua ◽  
Navpreet Ranu ◽  
Sarah E. Dilorio ◽  
Paul C. Blainey
Keyword(s):  
Genome Editing ◽  
Nuclease Activity ◽  
Single Strand ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Single Strand Break ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

ABSTRACTDNA single-strand breaks (SSBs), or ‘nicks’, are the most common form of DNA damage. Nicks occur at rates of tens of thousands per cell per day, and result from many sources including oxidative stress and endogenous enzyme activities. Accumulation of nicks, due to high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to learn how their locations and number affect cells, disease progression, and health outcomes. In addition to natural processes including DNA repair, leading genome-editing technologies rely on nuclease activity, including nick generation, at target sites. There is currently a pressing need for methods to study unintended nicking activity genome-wide to evaluate the impact of emerging genome editing tools on cells and organisms. Here we developed a new method, NickSeq, for efficient strand-specific profiling of nicks in complex DNA samples with single nucleotide resolution and low false-positive rates. NickSeq produces deep sequence datasets enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand. In this work, we apply NickSeq to profile off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. NickSeq will be useful in exploring the relevance of spontaneously occurring or repair-induced DNA breaks in human disease, DNA breaks caused by DNA damaging agents including therapeutics, and the activity of engineered nucleases in genome editing and other biotechnological applications.

scholarly journals Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Shuquan Rao ◽  
Yao Yao ◽  
Daniel E. Bauer
Keyword(s):  
Genome Editing ◽  
Genetic Variants ◽  
Association Studies ◽  
Genome Wide Association ◽  
Genome Wide Association Studies ◽  
Functional Studies ◽  
Functional Genetics ◽  
Genome Wide ◽  
Causal Variants ◽  
Experimental Approaches

AbstractGenome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.

scholarly journals Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

2019 ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A.M. Martin ◽  
Pierre Boudinot ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Cell Line ◽  
Genome Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Genetic Resistance ◽  
Model Organisms ◽  
Fish Cell ◽  
Genome Wide ◽  
Lentivirus Transduction

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

Genome editing's potential target diseases in the cardiovascular field

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Cardiovascular Diseases ◽  
Genome Editing ◽  
Target Cells ◽  
Clinical Use ◽  
Editing Efficiency ◽  
Potential Target ◽  
Genome Wide ◽  
Significant Barrier

Two types of cardiovascular diseases can be cured or prevented using genome editing. The liver is the organ that has received the most attention in terms of clinical genome editing for cardiovascular diseases. Off-target mutagenesis is a concern of any form of genome editing. Off-target mutations in target cells or tissues may lead to undesirable functional phenotypes, including cancer. For therapeutic editing of the heart, the authors claim it's critical to achieve high editing efficiency at a chosen genomic site in a desired tissue. Off-target editing can be tested genome-wide unbiasedly using newer cell-based methods. For low off-target impact, well-designed gRNAs are important. The delivery of genome editors to target tissues and cells is a significant barrier to clinical use.

Shaping the nebulous enhancer in the era of high-throughput assays and genome editing

2019 ◽  
Vol 21 (3) ◽  
pp. 836-850
Author(s):  
Edwin Yu-Kiu Ho ◽  
Qin Cao ◽  
Mengting Gu ◽  
Ricky Wai-Lun Chan ◽  
Qiong Wu ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Wide Spectrum ◽  
Data Interpretation ◽  
Future Studies ◽  
Transcriptional Enhancers ◽  
The Past ◽  
Genome Wide ◽  
Gray Areas ◽  
Practical Implications

Abstract Since the 1st discovery of transcriptional enhancers in 1981, their textbook definition has remained largely unchanged in the past 37 years. With the emergence of high-throughput assays and genome editing, which are switching the paradigm from bottom-up discovery and testing of individual enhancers to top-down profiling of enhancer activities genome-wide, it has become increasingly evidenced that this classical definition has left substantial gray areas in different aspects. Here we survey a representative set of recent research articles and report the definitions of enhancers they have adopted. The results reveal that a wide spectrum of definitions is used usually without the definition stated explicitly, which could lead to difficulties in data interpretation and downstream analyses. Based on these findings, we discuss the practical implications and suggestions for future studies.

scholarly journals In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

2018 ◽  
Author(s):  
Pinar Akcakaya ◽  
Maggie L. Bobbin ◽  
Jimmy A. Guo ◽  
Jose M. Lopez ◽  
M. Kendell Clement ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Strong Support ◽  
Guide Rna ◽  
Genome Wide ◽  
Highly Sensitive ◽  
Further Development ◽  
Target Effects ◽  
Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

scholarly journals A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa ◽  
Kuoyuan Cheng ◽  
Mark D. M. Leiserson ◽  
Prashant Jain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Types ◽  
Selective Advantage ◽  
Driver Mutations ◽  
Cancer Driver ◽  
Oncogenic Mutation ◽  
Genome Wide ◽  
Selection For ◽  
Mutant Cells ◽  
Selection Of

AbstractRecent studies have reported that genome editing by CRISPR–Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.

scholarly journals ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Camilo Breton ◽  
Peter M. Clark ◽  
Lili Wang ◽  
Jenny A. Greig ◽  
James M. Wilson
Keyword(s):  
Next Generation Sequencing ◽  
Genome Editing ◽  
Next Generation ◽  
Aav Vector ◽  
Genome Wide ◽  
A Genome ◽  
Vector Sequences ◽  
Target Activity ◽  
Generation Sequencing

Abstract Background Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism’s genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. Results Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. Conclusions In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.

scholarly journals Targeted control of toxin production by a mobile genetic element in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Emily Stevens ◽  
Daniel J. Morse ◽  
Dora Bonini ◽  
Seána Duggan ◽  
Tarcisio Brignoli ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nuclease Activity ◽  
Toxin Production ◽  
Helicase Domain ◽  
Human Pathogen ◽  
Genome Wide ◽  
A Genome ◽  
Invasive Diseases ◽  
Transcriptional Landscape ◽  
Integrative And Conjugative Element

Introductory ParagraphStreptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis1–3. Young children are at a particularly high risk, with an estimated 800,000 deaths worldwide in those under five attributable to invasive pneumococcal disease each year1–3. The cytolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, however, despite its importance to both the colonisation and pathogenic capabilities of this pathogen, the regulation of its expression is not well defined4–7. Using a genome-wide association approach we identified over a hundred potential affectors of Ply activity, including the Integrative and Conjugative Element (ICE) ICESp23FST818. This regulatory effect is mediated through the activity of a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like nuclease domains. The ZomB protein has potent ATP-dependent nuclease activity and binds specifically to the DNA containing a BOX repeat region that forms part of the ply operon. We hypothesise that with over 100 BOX regions across the pneumococcal genome, the acquisition of the zomB gene on ICESp23FST81 has the potential to re-wire the transcriptional landscape of this major human pathogen.

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