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More abundant and healthier meat: will the MSTN editing epitome empower the commercialization of gene editing in livestock?
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CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.
Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.
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Improving soybean fatty acid profiles by CRISPR/Cas12a-mediated gene editing of GmFAD2 and GmFAD3.
Using CRISPR/Cas12a, we engineered novel soybean germplasms by knocking out GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes, yielding elevated oleic or linoleic acid content. Soybean oil contains high levels of polyunsaturated fatty acids (PUFAs), which are known to reduce cholesterol levels and help prevent hypertension, thereby contributing significantly to human health. However, the chemical instability of PUFAs makes them susceptible to oxidation, a process that generates harmful trans-fatty acids. To address this issue, precise modulation of fatty acid composition in soybeans becomes critically important for health applications. In this study, we employed CRISPR/Cas12a gene editing technology to selectively knock out the GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes in soybean. This approach successfully created novel soybean germplasms with distinct fatty acid profiles: one with elevated oleic acid content and another with increased linoleic acid levels. These engineered variants provide valuable options for utilizing soybean oil with optimized fatty acid compositions tailored for specific health and nutritional purposes.
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Unleashing the potential: type I CRISPR-Cas systems in actinomycetes for genome editing.
Covering: up to the end of 2023Type I CRISPR-Cas systems are widely distributed, found in over 40% of bacteria and 80% of archaea. Among genome-sequenced actinomycetes (particularly Streptomyces spp.), 45.54% possess type I CRISPR-Cas systems. In comparison to widely used CRISPR systems like Cas9 or Cas12a, these endogenous CRISPR-Cas systems have significant advantages, including better compatibility, wide distribution, and ease of operation (since no exogenous Cas gene delivery is needed). Furthermore, type I CRISPR-Cas systems can simultaneously edit and regulate genes by adjusting the crRNA spacer length. Meanwhile, most actinomycetes are recalcitrant to genetic manipulation, hindering the discovery and engineering of natural products (NPs). The endogenous type I CRISPR-Cas systems in actinomycetes may offer a promising alternative to overcome these barriers. This review summarizes the challenges and recent advances in CRISPR-based genome engineering technologies for actinomycetes. It also presents and discusses how to establish and develop genome editing tools based on type I CRISPR-Cas systems in actinomycetes, with the aim of their future application in gene editing and the discovery of NPs in actinomycetes.
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CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement.
To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology-being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modified crops face. Developing transgenic-free edited plants without introducing foreign DNA has received regulatory approval in a number of countries. Several ongoing efforts from various countries are rapidly expanding to adopt the innovations. This review covers the mechanisms of CRISPR/Cas9, comparisons of CRISPR/Cas9 with other gene-editing technologies-including newly emerged Cas variants-and focuses on CRISPR/Cas9-targeted genes for rice crop improvement. We have further highlighted CRISPR/Cas9 vector construction model design and different bioinformatics tools for target site selection.
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CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists.
CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.
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CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy.
Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.
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Transgene-Free, Gene-Edited Cavendish Bananas (Musa acuminata, AAA).
Global consumer acceptance of gene-edited food crops is increasing with new breeding technologies that can modify the genome without foreign DNA integration. Here, we report an Agrobacterium-based system for transgene-free, gene editing of the banana cultivar, Cavendish. The protocol uses a three-tiered approach whereby cells containing T-DNA are enriched by positive antibiotic selection over a 48-h period, CRISPR/Cas9-mediated gene editing occurs over a short transient window and negative selection of cells containing T-DNA is achieved by the addition of 5-FC, which is converted to cytotoxic 5-FU by the co-expressed CODA enzyme. Two key enzymes in the carotenoid biosynthesis pathway were targeted as visual markers of editing: phytoene desaturase (pds) and lycopene β-cyclase (LCYb). Disruptive edits of the LCYb gene were easily identifiable at the calli stage of the regeneration process with cells appearing pink due to lycopene accumulation. Eight of 32 plants for pds and 34 of 125 plants for LCYb contained edits and were likely free of integrated vector DNA as determined by targeted genome sequencing and T-DNA-specific PCR, respectively. Plants containing disruptive tri-allelic edits in either gene displayed an albino phenotype. A subset of potentially transgene-free, edited plants was verified by whole genome sequencing. The transient editing protocol has an estimated 17.6% to 21.9% efficiency in generating plants containing disruptive tri-allelic modifications that are free of novel DNA. The protocol overcomes the regulatory limitations associated with the release of gene-edited, vegetatively propagated crops and provides an effective means of creating new disease-resistant and agronomically superior Cavendish cultivars.
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[Application of CRISPR/Cas9 Gene Editing Technique to Establish S1PR5 Gene Knockout Mice].
OBJECTIVE: To establish the S1PR5 gene knockout mouse model by using CRISPR/Cas9 gene editing technique so as to provide the tool for studying the regulating role of sphingosine-1-phosphate receptor 5 (S1PR5) in allogeneic hematopoietic stem cell transplantation. METHODS: Single guide RNA (sgRNA) plasmids against the exon 3 of S1PR5 were designed and constructed. Then the sgRNA and hCas9 were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6 mice. T7E1 digestion and gene sequencing were used to detect the mutations of S1PR5. Quantitative PCR (qPCR) and Western blot were used to detect the expression of S1PR5. RESULTS: Finally 2 kinds of F2 generation of homozygous S1PR5 deficent mice (S1PR5-170/-170 mice and S1PR5-215/-215 mice) were gained, and in these 2 model mice the S1PR5 did not express at mRNA and protein levels. CONCLUSION: A mouse model with S1PR5 dificiency has been successfully established, which shall lay a foundation for future investigation of S1PR5.
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CRISPR/Cas: A Successful Tool for Genome Editing in Animal Models.
CRISPR/Cas9 is an innovative molecular tool that is utilized in advanced biological applications. This review focuses on modifying the genomes of a wide range of animals by CRISPR/Cas9 for greater usability and higher efficiency, providing an overview of the function and mechanism of this system and the utilization of this system for medicinal research. The type II CRISPR-Cas system is found in the Enterobacteriaceae bacteria family, which uses this system as a defense against invading phages and plasmids. This system can be engineered to direct its action toward a targeted site for the modification of a specific genome.
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