High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus.
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ABSTRACT:
SUBMITTER: Nagalakshmi U
PROVIDER: S-EPMC9237674 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: Not available
| S-EPMC9342971 | biostudies-literature
Project description:Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single-guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al. (2021)1 and Aragonés et al. (2022).2.
| S-EPMC9943877 | biostudies-literature
Project description:A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
| S-EPMC3752207 | biostudies-literature
Project description:Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.
| S-EPMC4459934 | biostudies-literature
Project description:The use of CRISPR/Cas proteins for the creation of multiplex genome-engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR/Cas9 system for multiplex genome-engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome-editing in Arabidopsis thaliana. We identified optimized promoter sequences for driving expression of single transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome-engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome-editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex-genome-engineering in plant-based systems.
2021-09-15 | GSE162875 | GEO
Project description:Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
| S-EPMC8861562 | biostudies-literature
Project description:BackgroundFor ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. 'Chardonnay'). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy.ResultsBy comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. 'Chardonnay'. These results are directly correlated with Cas9 expression level.ConclusionsTaken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.
| S-EPMC10949784 | biostudies-literature
Project description:BackgroundApplication of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits.ResultsWe report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture.ConclusionsWe have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.
| S-EPMC6873407 | biostudies-literature
Project description:Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field.
| S-EPMC8070094 | biostudies-literature
Project description:CRISPR/Cas9, a bacterial adaptive immune system derived genome-editing technique, has become to be one of the most compelling topics in biotechnology. Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Here we reported highly efficient and multiplex genome editing in B. mori cell line and heritable site-directed mutagenesis of Bmku70, which is required for NHEJ pathway and also related to antigen diversity, telomere length maintenance and subtelomeric gene silencing, using CRISPR/Cas9 system. We established a simple and practicable method and obtained several Bmku70 knockout B. mori lines, and showed that the frequency of HR was increased in embryos of the Bmku70 knockout B. mori. The mutant lines obtained in this study could be a candidate genetic resource for efficient knock-in and fundamental research of DNA repair in B. mori. We also provided a strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect.
| S-EPMC3967148 | biostudies-literature