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Strategies and Protocols for Optimized Genome Editing in Potato.
The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.
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High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.
Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.
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Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.
Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.
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[Gene editing].
Development of the gene engineering techniques has raised worries that they will be used for construction of organism endangering humans and environment. In 1975 at the Asilomar conference, geneticists from many countries decided that genetic engineering brings more benefits than threats. In last years a new CRISPR-Cas technique emerged . It allows to make the precise changes in genomes, e.g. to inactivate particular genes or to replace mutated genes by their wild-type alleles. Inactivation in mice of genes corresponding to those whose mutations cause the genetic diseases in man allows to get model organisms for studying the etiology of given disease and for working out the methods of its curing. This technique can be applied for repairing genes whose mutations result in metabolic diseases and cancer. Some voices were raised that the technique can be potentially used for the "improvement" of man, what would create many ethical and social problems. Geneticists, ethicists and lawyers gathered in 2015 at the Washington conference, discussed these problems and proposed rules for their solving.
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A carrier-free multiplexed gene editing system applicable for suspension cells.
Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8+ T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8+ T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70-90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.
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Towards the elimination of infectious HPV: exploiting CRISPR/Cas innovations.
HPV has been conclusively associated with various human malignancies, making the development of prevention and treatment strategies for HPV-induced diseases a high priority. Currently, primary prevention methods include HPV immunization and routine screening, which significantly reduce the risk of HPV transmission. However, for patients diagnosed with invasive, advanced, or recurrent malignancies, non-virus-specific therapies frequently lead to drug resistance and adverse effects, resulting in minimal improvement in treatment efficacy for numerous patients. Viral genome-targeting therapy is emerging as a promising avenue for the future management of HPV infections. With the rapid advancement of genetic modification technologies, the CRISPR/Cas system has demonstrated significant potential in treating viral infections. Its ability to selectively target and edit viral genomes for elimination positions it as a highly effective approach for combating HPV. This review will explore the functions and applications of the CRISPR/Cas system as an innovative therapy for HPV. We will illustrate the prospective efficacy of CRISPR/Cas as a groundbreaking and promising cure for HPV infections, while also addressing the opportunities and challenges associated with this novel approach.
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Generation of a New Tau Knockout (tauΔex1) Line Using CRISPR/Cas9 Genome Editing in Mice.
Alzheimer's disease and other dementias present with tau pathology. Several mouse lines with knockout of the tau-encoding Mapt gene have been reported, yet findings often differed between lines and sites. Here, we report a new tau knockout strain (tauΔex1), generated by CRISPR/Cas9-mediated genome editing of intron -1/exon 1 of Mapt in C57Bl/6J mice. TauΔex1 mice had no overt phenotype, but, in line with previous models, they showed a significantly reduced susceptibility to excitotoxic seizures, with normal memory formation in young mice. This new in vivo resource will be made freely available to the research community.
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The novel insight into the outcomes of CRISPR/Cas9 editing intra- and inter-species.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeat technology/CRISPR-associated protein) is a widely used and powerful research tool in biosciences and a promising therapeutic agent for treating genetic diseases. Mutations induced by Cas9 are generally considered stochastic and unpredictable, thus hindering its applications where precise genetic alternations are required. Here, through deep sequencing and analysis of genome editing outcomes of multiple sites in four distinct species, we found that Cas9-induced mutations are coincident in mutation types but are significantly different in indel patterns among species. In human and mouse cells, indels were almost evenly distributed at both ends of the cleavage sites. However, the indels mainly appeared at the upstream of cleavage sites in Bombyx mori, while they predominantly occurred downstream of the cleavage sites in the zebrafish Danio rerio. We also found that within a species, indel patterns are sequence dependent, wherein deletions between two adjacent micro-homology sequences were the most frequently observed mutations in the repair spectrum. These results suggested the species differences in DNA repair processes during Cas9-induced gene editing, and the important role of sequence structure at the target site in predicting the gene editing outcome.
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[CRISPR-Cas9 editing of HIV-1 neutralizing human B cells].
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Genome Editing for Developing Disease-Resistant Plants.
Genome editing tools, most notably the CRISPR-Cas9 system, have transformed modern crop breeding by allowing precise and efficient modification of plant genomes, particularly for enhancing resistance to disease. In this chapter, we present two major CRISPR-based approaches that have been successfully applied to develop disease-resistant crops: (1) gene knockout of host susceptibility (S) genes to block pathogen compatibility, and (2) targeted insertion of resistance alleles or synthetic genes using the homology-directed repair (HDR) pathway. Both strategies utilize the specificity and efficiency of CRISPR-Cas9 to introduce defined edits in crops such as rice. This chapter outlines comprehensive step-by-step protocols covering key elements of the process, including target site selection, construction of sgRNA and Cas9 vectors, plant transformation methods, and mutation validation techniques. Together, these methods provide a powerful platform for engineering plant immune responses and offer practical tools to accelerate the breeding of disease-resistant cultivars.
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