SUMMARY
Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR‐associated proteins (Cas) have revolutionized genome editing in many organisms, including ...plants. Most CRISPR‐Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target‐specific and a screening process is usually required to identify those most effective. Plant virus‐derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus‐induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX‐based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus‐free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR‐Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
Significance Statement
Plant virus‐derived vectors allow fast and efficient delivery of synthetic guide RNAs into adult plants for CRISPR‐Cas‐based genome editing. We engineered a Potato virus X vector for CRISPR‐Cas genome editing of solanaceous plants. This vector expresses unspaced arrays of synthetic guide RNAs and achieves multiplex editing in adult plant tissues in a few days. Virus‐free multiplex genome‐edited plants with biallelic mutations can be easily obtained from inoculated plants.
Cell therapy has the potential to revolutionize many areas of unmet clinical needs including cancer, degenerative and metabolic diseases, genetic disorders, infection, inflammation and autoimmunity. ...Both RNA-based and genomic editing technologies (e.g. CRISPR/Cas9) are actively investigated to design cellular therapies with customized functionalities. One of the key advantages of these new technologies is their highly adaptable nature that enables sophisticated cellular engineering, rational design and rapid pursuit of new therapeutic targets.
At Portal, we have developed a gentle silicon membrane-based delivery technology that facilitates mechanoporation of cells and can be easily integrated with existing clinical-scale workflows. Our results in primary immune cells using monocytes, T, B and NK cells demonstrate over 80% efficiency and viability of cellular engineering. CRISPR/Cas9 RNPs, mRNAs and siRNAs have been successfully delivered as single cargos or in a multiplexed fashion, demonstrating the potential for either single- or multi-step cell engineering workflows which can address several targets. Furthermore, data in iPSCs and HSCs have also shown significant potential in stem cell engineering and differentiation capabilities. This technology is currently being implemented for research and clinical scale use - including in an integrated Point-of-Care manufacturing system. With continued progress in simplifying cell engineering using both RNA-based and genomic editing technologies, we aim to unlock vast biological potential in the field of cell therapy while simultaneously reducing or even eliminating safety or manufacturing concerns such as inadvertent genome alteration associated with other delivery modalities such as lentiviral vectors.
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) genome editing system is a powerful tool for targeted gene modifications in a wide range of ...species, including plants. Over the last few years, this system has revolutionized the way scientists perform genetic studies and crop breeding, due to its simplicity, flexibility, consistency and high efficiency. Considerable progress has been made in optimizing CRISPR/Cas9 systems in plants, particularly for targeted gene mutagenesis. However, there are still a number of important challenges ahead, including methods for the efficient delivery of CRISPR and other editing tools to most plants, and more effective strategies for sequence knock-ins and replacements. We provide our viewpoint on the goals, potential concerns and future challenges for the development and application of plant genome editing tools.
Advances in technology have led to a large increase in the number of plant organellar sequences, but sampling remains uneven.Most land-plant mitogenomes are difficult to assemble accurately due to ...alternative structural configurations and interference from transferred sequences.Pan-organellar genomes have provided new evolutionary insights and have provided targets for editing to study changes in gene function during lineage divergence.Organellar genome editing – such as mitochondrially targeted transcription activator-like effector nucleases (TALENs) and double-stranded DNA-specific cytidine deaminase (DddA)-derived cytosine base editors – enables the functional verification of open reading frames (ORFs) and non-coding regions.Plastid transformation has been used in an array of modifications – such as to improve photosynthetic efficiency, and the biosynthesis of vaccines – while mitochondrial transformation lags far behind due to technical challenges.
Plastids and mitochondria are the only organelles that possess genomes of endosymbiotic origin. In recent decades, advances in sequencing technologies have contributed to a meteoric rise in the number of published organellar genomes, and have revealed greatly divergent evolutionary trajectories. In this review, we quantify the abundance and distribution of sequenced plant organellar genomes across the plant tree of life. We compare numerous genomic features between the two organellar genomes, with an emphasis on evolutionary trajectories, transfers, the current state of organellar genome editing by transcriptional activator-like effector nucleases (TALENs), transcription activator-like effector (TALE)-mediated deaminase, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), as well as genetic transformation. Finally, we propose future research to understand these different evolutionary trajectories, and genome-editing strategies to promote functional studies and eventually improve organellar genomes.
Plastids and mitochondria are the only organelles that possess genomes of endosymbiotic origin. In recent decades, advances in sequencing technologies have contributed to a meteoric rise in the number of published organellar genomes, and have revealed greatly divergent evolutionary trajectories. In this review, we quantify the abundance and distribution of sequenced plant organellar genomes across the plant tree of life. We compare numerous genomic features between the two organellar genomes, with an emphasis on evolutionary trajectories, transfers, the current state of organellar genome editing by transcriptional activator-like effector nucleases (TALENs), transcription activator-like effector (TALE)-mediated deaminase, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), as well as genetic transformation. Finally, we propose future research to understand these different evolutionary trajectories, and genome-editing strategies to promote functional studies and eventually improve organellar genomes.
The mud loach (Misgurnus anguillicaudatus) is one of the most important aquaculture fishes in Asia. The application of the CRISPR/Cas9 system in the cultivation of loach strains with valuable ...economic traits presents great potential. Myostatin is a candidate factor that can be used for the cultivation of fast-growing loach strains. In this study, the appropriate microinjection dose of the Cas9 protein (600 pg) for genome editing was determined considering both the mutation rate and survival rate of loach embryos. With this microinjection dose of the Cas9 protein, both the exogenous GFP and endogenous myostatin genes were efficiently edited using the CRISPR/Cas9 system. Compared with that in wild-type siblings, the average body weight of the fish in 1-, 2- and 3-month-old myostatin mutant chimeric loaches was significantly increased by 34.9% (P < 0.001), 15.5% (P = 0.024) and 27.8% (P = 0.012), respectively. Mutation of mstn significantly increased the number of muscle fibers and total area of muscle fibers. Also, lipid accumulation was significantly higher in chimeric mutant loach. Additionally, the mRNA levels of myogenesis (myod and myog) and lipogenesis-related genes (scd, fabp1, fabp2 and dgat) were significantly upregulated in chimeric mutants. In particular, the mRNA level of the stearoyl-CoA desaturase gene, whose product catalyses the formation of monounsaturated fatty acids from saturated fatty acids, was increased by 23-fold (P = 0.034) and 736-fold (P < 0.05) in 3 dpf and 1-month-old chimeric loaches, respectively. The gene expression results indicated that the targeted disruption of myostatin activated myogenesis and adipogenesis in loach. Our study is highly valuable for cultivating fast-growing loach strains and understanding the specific role of myostatin in lipogenesis.
•The appropriate microinjection dose of the Cas9 protein for genome editing was determined in loach Misgurnus anguillicaudatus.•Fast-growing loaches were obtained through the targeted mutation of myostatin.•Loss of myostatin activated myogenesis in loach.•Loss of myostatin activated adipogenesis in loach.
Summary
Gene‐editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range ...of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene‐editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene‐editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream ‘omics’ technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or ‘non‐targeted metabolite analysis’ needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of ‘omics’ technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops.
Significance Statement
Unlike with transgenic approaches, it is not facile to detect gene‐editing events forensically at the molecular level, as no foreign DNA exists in gene‐edited lines. The limitation in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process itself. Here, we highlight the use of metabolomics as a means to characterize (and in some cases identify) gene‐edited material and champion its use for regulatory purposes.
Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues ...of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a “safe harbor” site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model. Three dose levels of recombinant AAV2/8 vectors encoding a pair of ZFNs and a hIDS cDNA donor were administered systemically in MPS II mice. Supraphysiological, vector dose-dependent levels of IDS enzyme were observed in the circulation and peripheral organs of ZFN+donor-treated mice. GAG contents were markedly reduced in tissues from all ZFN+donor-treated groups. Surprisingly, we also demonstrate that ZFN-mediated genome editing prevented the development of neurocognitive deficit in young MPS II mice (6–9 weeks old) treated at high vector dose levels. We conclude that this ZFN-based platform for expression of therapeutic proteins from the albumin locus is a promising approach for treatment of MPS II and other lysosomal diseases.
AAV-mediated in vivo delivery of ZFN and IDS donor resulted in site-specific gene insertion and dose-dependent IDS expression in a mouse model of MPS II. These results support a currently open clinical trial, the first ever in vivo human genome editing study to be conducted.
Genome editing technologies are promising for conventional mutagenesis breeding, which takes a long time to remove unnecessary mutations through backcrossing and create new lines because they ...directly modify the target genes of elite strains. In particular, this technology has advantages for traits caused by the loss of function. Many efforts have been made to utilize this technique to introduce valuable features into crops, including maize, soybeans, and tomatoes. Several genome-edited crops have already been commercialized in the US and Japan. Melons are an important vegetable crop worldwide, produced and used in various areas. Therefore, many breeding efforts have been made to improve its fruit quality, resistance to plant diseases, and stress tolerance. Quantitative trait loci (QTL) analysis was performed, and various genes related to important traits were identified. Recently, several studies have shown that the CRISPR/Cas9 system can be applied to melons, resulting in its possible utilization as a breeding technique. Focusing on two productivity-related traits, disease resistance, and fruit quality, this review introduces the progress in genetics, examples of melon breeding through genome editing, improvements required for breeding applications, and the possibilities of genome editing in melon breeding.
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