scholarly journals Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA

2020 ◽  
Vol 185 ◽  
pp. 04018
Author(s):  
Calvin Guo ◽  
David Zhen
Keyword(s):  
Genome Editing ◽  
Life Science ◽  
Genetic Diseases ◽  
Science Research ◽  
New Drugs ◽  
Limiting Factor ◽  
Widespread Application ◽  
Genetic Characteristics ◽  
Target Sites ◽  
Target Effect

CRISPR/Cas9 genome editing technology is the frontier of life science research. They have been used to cure human genetic diseases, achieve cell personalized treatment, develop new drugs, and improve the genetic characteristics of crops and other fields. This system relies on the enzyme Cas9 cutting target DNA (on target) under the guidance of sgRNA, but it can also cut non-target sites, which results in offtarget effects, thus causing uncontrollable mutations. The risk of off-target effect in CRISPR technology is the main limiting factor that affects the widespread application of CRISPR technology. How to evaluate and reduce the off-target effect is the urgent problem to be solved. In this work, we build up a model that can predict the score of being off-target. Through comparison with the complete genome of the target and precise mathematics that calculate the potential risk of being off-target, we optimize the sgRNA, which is capable of reducing the off-target effect. The result has proven that we can efficiently and quickly identify and screen the best editing target sites with our model. The CRISPR/Cas9 system, not even being perfected yet, has already demonstrated its potential in the field of genome editing. Hopefully through our model, the CRISPR/Cas9 system can quickly apply to more branches in life science and cure those diseases that have been previously incurable.

scholarly journals Gene Editing and Genome Engineering with CRISPR-Cas9

2017 ◽  
Vol 01 (02) ◽  
pp. 99-107 ◽  
Author(s):  
Emmanuelle Charpentier
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
Life Science ◽  
Science Research ◽  
Cell Types ◽  
Life Science Research ◽  
Domains Of Life ◽  
Modulation Of Gene Expression

The RNA-programmable CRISPR-Cas9 technology allows precise and efficient engineering or correction of mutations, modulation of gene expression and marking of DNA in a wide variety of cell types and organisms in the three domains of life. Because of its versatility and ease of design, this powerful technology has been rapidly and universally adopted for genome editing applications in life science research. It is also recognized for its promising and potentially transformative applications in biotechnology, medicine and agriculture.

scholarly journals Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianhang Wang ◽  
Mingxing Tu ◽  
Ya Wang ◽  
Wuchen Yin ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Editing ◽  
Genome Sequencing ◽  
Plant Biotechnology ◽  
High Specificity ◽  
Fruit Trees ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Indel Mutation ◽  
Target Sites

AbstractThe CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

scholarly journals Genome editing: A breakthrough in life science and medicine [Review]

2016 ◽  
Vol 63 (2) ◽  
pp. 105-110 ◽  
Author(s):  
Izuho Hatada ◽  
Takuro Horii
Keyword(s):  
Genome Editing ◽  
Life Science ◽  
Medicine Review

The ethics of genome editing in the clinic: A dose of realism for healthcare leaders

2017 ◽  
Vol 30 (3) ◽  
pp. 159-163
Author(s):  
Tania Bubela ◽  
Yael Mansour ◽  
Dianne Nicol
Keyword(s):  
Genome Editing ◽  
Germ Line ◽  
Genetic Diseases ◽  
Somatic Cells ◽  
Clinical Translation ◽  
Preclinical Research ◽  
Societal Issues ◽  
Realistic Assessment ◽  
Healthcare Leaders

Genome editing technologies promise therapeutic advances for genetic diseases. We discuss the ethical and societal issues raised by these technologies, including their use in preclinical research, their potential to address mutations in somatic cells, and their potential to make germ line alterations that may be passed to subsequent generations. We call for a proportionate response from health leaders based on a realistic assessment of benefits, risks, and timelines for clinical translation.

Conferring a Federal Property Right in Genetic Material: Stepping into the Future with the Genetic Privacy Act

1996 ◽  
Vol 22 (1) ◽  
pp. 109-134
Author(s):  
Michael M.J. Lin
Keyword(s):  
Ethical Issues ◽  
Raw Materials ◽  
Genetic Diseases ◽  
Genetic Material ◽  
Genetic Privacy ◽  
Genetic Characteristics ◽  
Dna Evidence ◽  
Privacy Act ◽  
National Rule ◽  
Social And Ethical Issues

“A wise man can hear profit in the wind.”—Pel, quoting the Ferengi Rules of AcquisitionThe expansive biotechnology field includes many facets of medical research, from drug discovery and design, to gene therapy and the diagnosis of genetic diseases, to the use of deoxyribonucleic acid (DNA) evidence to identify individuals and genetic characteristics. The biotechnology industry requires a readily available supply of biological raw materials; much of current research is founded on cells, tissues, organs, fetal tissues and placentas, and other samples derived from human donors. However, this growing need for raw materials presents many economic, social, and ethical issues to society, researchers, and the existing legal regime. Furthermore, because courts and legislatures fail to provide a clear national rule regarding biological materials, the resulting legal uncertainties chill research and investment. Although very few cases address property rights in a person’s organs, tissues, and genetic material, the issues of autonomy and privacy involved evoke analogies to deep-seated issues such as slavery, the freezing of embryos, and abortion.

External Stimuli-Responsive Nanoparticles for Spatially and Temporary Controlled Delivery of CRISPR-Cas Genome Editors

2021 ◽  
Author(s):  
Ruosen Xie ◽  
Yuyuan Wang ◽  
Shaoqin Gong
Keyword(s):  
Genome Editing ◽  
Genetic Diseases ◽  
Delivery Systems ◽  
Controlled Delivery ◽  
Stimuli Responsive ◽  
New Therapies ◽  
External Stimuli

The CRISPR–Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. Up to date, various viral and non-viral delivery systems have...

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

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