scholarly journals Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Open Biology ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Yile Hao ◽  
Qinhua Wang ◽  
Jie Li ◽  
Shihui Yang ◽  
Yanli Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
High Efficiency ◽  
Helicase Domain ◽  
Type I ◽  
Strand Breaks ◽  
Gene Insertion ◽  
Type V

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

scholarly journals Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

2021 ◽  
Author(s):  
Yile Hao ◽  
Qinhua Wang ◽  
Jie Li ◽  
Shihui Yang ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
High Efficiency ◽  
Double Strand Breaks ◽  
Helicase Domain ◽  
Type I ◽  
Strand Breaks ◽  
Type V

New CRISPR-based genome editing technologies are developed to continuedly drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon an endogenous Type I system of Zymomonas mobilis. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity; an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing, without visible cell killing. By harnessing this CRISPR-nCas3, deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, easy, yet useful approach to convert many endogenous Type I systems into advanced genome editing tools. We envision that many CRISPR-nCas3-based toolkits would be soon available for various industrially important non-model bacteria that carry active Type I systems to facilitate high-throughput prokaryotic engineering.

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals Enabling large-scale genome editing by reducing DNA nicking

2019 ◽  
Author(s):  
Cory J. Smith ◽  
Oscar Castanon ◽  
Khaled Said ◽  
Verena Volf ◽  
Parastoo Khoshakhlagh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Single Strand Breaks ◽  
Dna Nicking ◽  
Mammalian Genomes ◽  
Induced Pluripotent

AbstractTo extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

scholarly journals Retrons and their applications in genome engineering

2019 ◽  
Vol 47 (21) ◽  
pp. 11007-11019 ◽  
Author(s):  
Anna J Simon ◽  
Andrew D Ellington ◽  
Ilya J Finkelstein
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Double Strand Breaks ◽  
Modern Biology ◽  
Genetic Transmission ◽  
Strand Breaks ◽  
Reverse Transcriptases ◽  
Natural Function ◽  
And Function

Abstract Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as bacteriophage lambda proteins for recombineering and CRISPR nucleases for eliciting site-specific double-strand breaks. Less well-known is a widely distributed class of bacterial retroelements, retrons, that employ specialized reverse transcriptases to produce noncoding intracellular DNAs. Retrons’ natural function and mechanism of genetic transmission have remained enigmatic. However, recent studies have harnessed their ability to produce DNA in situ for genome editing and evolution. This review describes retron biology and function in both natural and synthetic contexts. We also highlight areas that require further study to advance retron-based precision genome editing platforms.

scholarly journals Enabling large-scale genome editing at repetitive elements by reducing DNA nicking

2020 ◽  
Vol 48 (9) ◽  
pp. 5183-5195 ◽  
Author(s):  
Cory J Smith ◽  
Oscar Castanon ◽  
Khaled Said ◽  
Verena Volf ◽  
Parastoo Khoshakhlagh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Repetitive Elements ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Single Strand Breaks ◽  
Dna Nicking ◽  
Induced Pluripotent

Abstract To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements—ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

scholarly journals Characterization and repurposing of the endogenous Type I-F CRISPR–Cas system of Zymomonas mobilis for genome engineering

2019 ◽  
Vol 47 (21) ◽  
pp. 11461-11475 ◽  
Author(s):  
Yanli Zheng ◽  
Jiamei Han ◽  
Baiyang Wang ◽  
Xiaoyun Hu ◽  
Runxia Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Functional Characterization ◽  
Type I ◽  
Dna Targeting ◽  
Engineering Practices ◽  
Restriction Modification ◽  
Shed Light

Abstract Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR–Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction–modification (R–M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R–M system. This study might shed light on exploiting and improving CRISPR–Cas systems in other microorganisms for genome editing and metabolic engineering practices.

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.

Novel Materials for Myco-Decontamination of Cyanide-Containing Wastewaters through Microbial Biotechnology

2021 ◽  
Vol 1037 ◽  
pp. 751-758
Author(s):  
Igor N. Pavlov ◽  
Yulia A. Litovka
Keyword(s):  
Large Scale ◽  
High Efficiency ◽  
Pine Sawdust ◽  
Microbial Biotechnology ◽  
Tailing Dam ◽  
Large Scale Cultivation ◽  
Siberian Pine ◽  
Uniform Ratio

This study examined the effectiveness of decontamination of industrial cyanide-containing water using mycelium-based lignocellulosic materials. These results suggest that fungi biomass and plant substrates can be used successfully in the treatment of wastewater contaminated by cyanide. Fungi were isolated from old wood samples taken from a tailing dam with high cyanide content (more than 20 years in semi-submerged condition). All isolated fungi belonged to the genus Fusarium. Fusarium oxysporum Schltdl. is most effective for biodegradation of cyanide-containing wastewaters (even at low temperatures). The most optimal lignocellulosic composition for production of mycelium-based biomaterial for biodegradation of cyanide wastewater consists of a uniform ratio of Siberian pine sawdust and wheat straw. The high efficiency of mycelium-based materials has been experimentally proven in vitro at 15-25 ° C. New fungal biomaterials are provide decrease in the concentration of cyanide ions to 79% (P <0.001). Large-scale cultivation of fungi biomass was carried out by the periodic liquid-phase cultivation. The submerged biomass from bioreactor was used as an inoculum for the production of mycelium-based materials for bioremediation of cyanide wastewater in situ (gold mine tailing).

scholarly journals A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus

2019 ◽  
Vol 48 (1) ◽  
pp. 472-485 ◽  
Author(s):  
Felix Lansing ◽  
Maciej Paszkowski-Rogacz ◽  
Lukas Theo Schmitt ◽  
Paul Martin Schneider ◽  
Teresa Rojo Romanos ◽  
...  
Keyword(s):  
Human Genome ◽  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Genomic Sequence ◽  
Safe Alternative ◽  
Protein Coding ◽  
Dna Binding Specificity ◽  
Human Genomic ◽  
Human Genomic Sequence

Abstract Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

scholarly journals Single step, high efficiency CRISPR-Cas9 genome editing in primary human disease-derived fibroblasts

2018 ◽  
Author(s):  
Matteo Martufi ◽  
Robert B. Good ◽  
Radu Rapiteanu ◽  
Tobias Schmidt ◽  
Eleni Patili ◽  
...  
Keyword(s):  
Genome Editing ◽  
Drug Targets ◽  
Type I Collagen ◽  
High Efficiency ◽  
Cell Types ◽  
Single Step ◽  
Lung Fibroblasts ◽  
Type I ◽  
Physiological Relevance ◽  
Low Passage

AbstractGenome editing is a tool that has many applications including the validation of potential drug targets. However, performing genome editing in low passage, primary human cells with the greatest physiological relevance, is notoriously difficult. High editing efficiency is desired because it enables gene knock outs (KO) to be generated in bulk cellular populations and circumvents the problem of having to generate clonal cell isolates. Here, we describe a single step workflow enabling >90% KO generation in primary human lung fibroblasts via CRISPR ribonucleoprotein delivery, in the absence of antibiotic selection or clonal expansion. As proof of concept, we performed a disease relevant phenotypic assay measuring collagen deposition in response to TGFβ and demonstrated SMAD3 but not SMAD2 dependent deposition of type I collagen following knockout of each using our single step methodology. The optimization of this workflow can readily be transferred to other primary cell types.

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