Endogenous Hydrogen Peroxide Can Efficiently Regulate CRISPR-Cas9 Based Gene Editing

2022 ◽  
Author(s):  
Jizhong Zhao ◽  
Hongmei Hu ◽  
Jingwen Zhang ◽  
Yuanyuan Li ◽  
Li Wang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Genome Editing ◽  
Gene Editing ◽  
Life Science ◽  
Plant Research

CRISPR-Cas9 genome editing technology is an emerging tool in life science, clinical and plant research. The technique offers a remarkable specificity and efficiency in gene editing. In this study, we...

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 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 In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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

scholarly journals Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

2021 ◽  
Author(s):  
Xiaoen Huang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species ◽  
Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

scholarly journals Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3993-3993
Author(s):  
Linda Yingqi Lin ◽  
Samuele Cancellieri ◽  
Jing Zeng ◽  
Francesco Masillo ◽  
My Anh Nguyen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Clinical Trials ◽  
Genome Editing ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
African Ancestry ◽  
Great Promise ◽  
Personal Genome ◽  
Hematopoietic Stem ◽  
The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genome editing in large animals: current status and future prospects

2019 ◽  
Vol 6 (3) ◽  
pp. 402-420 ◽  
Author(s):  
Jianguo Zhao ◽  
Liangxue Lai ◽  
Weizhi Ji ◽  
Qi Zhou
Keyword(s):  
Regenerative Medicine ◽  
Genome Editing ◽  
Biomedical Research ◽  
Human Disease ◽  
Gene Editing ◽  
Disease Modeling ◽  
Current Status ◽  
Future Prospects ◽  
Recent Advances ◽  
Large Animals

AbstractLarge animals (non-human primates, livestock and dogs) are playing important roles in biomedical research, and large livestock animals serve as important sources of meat and milk. The recently developed programmable DNA nucleases have revolutionized the generation of gene-modified large animals that are used for biological and biomedical research. In this review, we briefly introduce the recent advances in nuclease-meditated gene editing tools, and we outline these editing tools’ applications in human disease modeling, regenerative medicine and agriculture. Additionally, we provide perspectives regarding the challenges and prospects of the new genome editing technology.

scholarly journals 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

scholarly journals Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vijaydev Ganesan ◽  
Michael Spagnuolo ◽  
Ayushi Agrawal ◽  
Spencer Smith ◽  
Difeng Gao ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Gene Editing ◽  
Molecular Genetic ◽  
Industrial Applications ◽  
Cell Factory ◽  
Fuel Feed ◽  
Renewable Chemicals ◽  
Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

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