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Functional genomic tools for emerging model species.
Most studies in the field of ecology and evolution aiming to connect genotype to phenotype rarely validate identified loci using functional tools. Recent developments in RNA interference (RNAi) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas genome editing have dramatically increased the feasibility of functional validation. However, these methods come with specific challenges when applied to emerging model organisms, including limited spatial control of gene silencing, low knock-in efficiencies, and low throughput of functional validation. Moreover, many functional studies to date do not recapitulate ecologically relevant variation, and this limits their scope for deeper insights into evolutionary processes. We therefore argue that increased use of gene editing by allelic replacement through homology-directed repair (HDR) would greatly benefit the field of ecology and evolution.
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Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica.
Yarrowia 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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Generation of Functional Genetic Study Models in Zebrafish Using CRISPR-Cas9.
CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.
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Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9.
Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.
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Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants.
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[Adapting Mouse Genome Editing Technique from Scratch Using in utero Electroporation].
Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology to develop an easy-to-use and cost-effective pipeline enabling mouse genome-editing from scratch with minimal requirements to operator skills and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce Il10 knockout in C57BL/6 mice. Three animals out of 13 delivered pups (23%) were genetically compromised at Il10 locus suggesting the feasibility of the approach. This protocol provides detailed description of the used technical settings paired with troubleshooting tips and could be of interest to those who aim at establishing in-house mouse transgenesis pipeline at minimal equipment cost from scratch.
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CRISPR meets AI-based robotics: Advancing sustainable agriculture.
In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.
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CRISPR/Cas9.
CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.
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Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR.
CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T1 generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T2 mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.
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Multigene editing: current approaches and beyond.
CRISPR/Cas9 multigene editing is an active and widely studied topic in the fields of biomedicine and biology. It involves a simultaneous participation of multiple single-guide RNAs (sgRNAs) to edit multiple target genes in a way that each gene is edited by one of these sgRNAs. There are possibly numerous sgRNA candidates capable of on-target editing on each of these genes with various efficiencies. Meanwhile, each of these sgRNA candidates may cause unwanted off-target editing at many other genes. Therefore, selection optimization of these multiple sgRNAs is demanded so as to minimize the number of sgRNAs and thus reduce the collective negative effects caused by the off-target editing. This survey reviews wet-laboratory approaches to the implementation of multigene editing and their needs of computational tools for better design. We found that though off-target editing is unavoidable during the gene editing, those disfavored cuttings by some target genes' sgRNAs can potentially become on-target editing sites for some other genes of interests. This off-to-on role conversion is beneficial to optimize the sgRNA selection in multigene editing. We present a preference cutting score to assess those beneficial off-target cutting sites, which have a few mismatches with their host genes' on-target editing sites. These potential sgRNAs can be prioritized for recommendation via ranking their on-target average cutting efficiency, the total off-target site number and their average preference cutting score. We also present case studies on cancer-associated genes to demonstrate tremendous usefulness of the new method.
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