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Genome editing creates disease-resistant crops without yield penalties.
Application of disease-resistant varieties is the most effective and environmentally friendly way to control crop diseases. However, there is often a trade-off between disease resistance and yield. Several recent studies have demonstrated that genome-editing technology brings a new strategy for generating disease-resistant crops without yield penalties.
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Attaining the promise of plant gene editing at scale.
Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.
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CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.
The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.
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An engineered CRISPR-Cas12i tool for efficient multiplexed genome editing.
Developing efficient and simplified tools for multiplexed genome editing remains challenging due to limitations in precursor CRISPR RNA (pre-crRNA) processing and reliance on additional RNA-based regulatory components. Cas12i.3, a small RNA-guided nuclease, reportedly lacks pre-crRNA processing ability, restricting its multiplexing capability. Here, we engineered Cas12i.3 by optimizing CRISPR RNA (crRNA) design, codon usage, and exonuclease fusion, generating initial optimized Cas12i (IOCas12i) system. Further rational design and amino acid mutations yielded the highly efficient enhanced optimized Cas12i (EOCas12i) systems, EOCas12i-Combo1 and EOCas12i-Combo2, exhibiting 2.5- to 22.8-fold and 3.0- to 60.0-fold editing efficiencies relative to wild-type Cas12i.3, comparable to Streptococcus pyogenes Cas9 (SpCas9) and Lachnospiraceae bacterium Cas12a (LbCas12a). Additionally, they exhibited high specificity and produced longer insertions and deletions (indels) that may facilitate gene knockout. Notably, both variants enabled efficient multiplexed editing of up to 30 targets using compact crRNA arrays. These advancements position EOCas12i-Combo1 and EOCas12i-Combo2 as promising platforms for multiplexed genome editing applications.
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Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells.
Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.
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[Advances in the application strategies of CRISPR/Cas9 technology in chimeric antigen receptor T cell therapy for hematological malignancies].
Chimeric antigen receptor (CAR) T-cell therapy has achieved breakthroughs in treating relapsed/refractory B-cell malignancies. However, it still faces challenges, including complex manufacturing processes, limited indications, T-cell exhaustion, and insufficient durability of therapeutic efficacy. CRISPR/Cas9, a highly efficient and relatively simple gene-editing technology, offers new avenues for overcoming these limitations. This review briefly outlines the working mechanism of CRISPR/Cas9 and focuses on its recent applications and clinical practices in developing universal CAR T-cells, enhancing T-cell function, and extending CAR T-cell therapy to T-cell and myeloid leukemias. Furthermore, this review highlights optimization strategies developed over the past two years to enhance the editing precision, delivery efficiency, and safety of the CRISPR/Cas9 system, aiming to provide insights for the optimal design and clinical application of CAR T-cell therapy.
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Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects.
Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.
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Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.
This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).
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A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards.
Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR-Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.
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Roles of Mitochondrial DNA Signaling in Immune Responses.
Mitochondrial DNA (mtDNA) plays an important role in immune responses during the evolution. The present chapter systemically describes its role on immune-related diseases and its interaction on immune responses. It is important to explore the main function and mechanisms of mtDNA in immune responses by which mtDNA regulates the signaling pathways of Toll-like receptor 9, autophagy, and STING. There are potentials to discover therapeutic targets of mtDNA in immune diseases and inflammation. It will be more exciting if the CRISPR-Cas9 method can be applied for mtDNA gene editing to cure diseases and provide a novel insight of mtDNA in immune responses as well as new therapies.
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