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Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.
Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.
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A CRISPR view on genetic screens in Toxoplasma gondii.
Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety of organisms, including unicellular parasites. Today, the CRISPR-Cas9 technology is vastly applied in high-throughput screens to investigate interactions between the Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro and in vivo T. gondii screens performed in naive and restrictive conditions have led to the discovery of essential and fitness-conferring T. gondii genes, as well as factors important for virulence and dissemination. Recent studies have adapted the CRISPR-Cas9 screening technology to study T. gondii genes based on phenotypes unrelated to parasite survival. These advances were achieved by using conditional systems coupled with imaging, as well as single-cell RNA sequencing and phenotypic selection. Here, we review the state-of-the-art of CRISPR-Cas9 screening technologies with a focus on T. gondii, highlighting strengths, current limitations and future avenues for its development, including its application to other Apicomplexan species.
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Anything impossible with CRISPR/Cas9?
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Genome Editing by CRISPR/Cas9 in Polyploids.
CRISPR/Cas system has been widely used for genome editing in the past few years. Even though it has been performed in many polyploid species to date, its efficient accomplishment in these organisms is still a challenge. The presence of multiple homoeologous genes as targets for their editing requires more rigorous work and specific needs to assess successful genome editing. Here, we describe a general stepwise protocol to select target sites, design sgRNAs, indicate vector requirements, and screen CRISPR/Cas9-mediated genome editing in polyploid species.
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Evaluation of cucumber UBL5 promoter as a tool for transgene expression and genome editing in plants.
Transgene expression and genome editing can help improve cucumber varieties to better respond to climate change. This study aimed to evaluate the applicability of the CsUBL5 promoter in transgene expression and genome editing in cucumber. The CsUBL5 promoter was cloned and analyzed to identify cis-elements that respond to abiotic signals, hormones, signal molecules, and nutrient treatments. 5' deletion constructs of the promoter were tested for their ability to drive GUS reporter expression in cucumber cotyledons, Arabidopsis seedlings, and tobacco leaves, and their response to various treatments including SA, light, drought, IAA, and GA was determined. The results showed that the CsUBL5 promoter effectively drove transgene expression in these plants, and their expressions under treatments were consistent with the predicted cis-elements, with some exceptions. Furthermore, the pCsUBL5-749 deletion construct can improve genome editing efficiency in cucumber when driving Cas9 expression. The editing efficiency of two sgRNAs targeting the ATG6 gene in cucumber was up to 4.6-fold higher using pCsUBL5-749 compared to a rice UBI promoter, although the effects of changing promoter on the editing efficiency is sgRNA specific. These findings highlight the potential utility of the CsUBL5 promoter for improving cucumber varieties through genetic engineering and genome editing. It also demonstrates the importance of modulating Cas9 expression to increase genome editing efficiency in cucumbers.
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Advances toward Curing HIV-1 Infection in Tissue Reservoirs.
A disease of more than 39.6 million people worldwide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts toward a sterilizing or functional cure. On the horizon is research concentrated in multiple separate but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including checkpoint inhibitors, and gene editing. Here, we review the concept of targeting the HIV-1 tissue reservoirs, with an emphasis on the central nervous system, and describe relevant new work in functional cure research and strategies for HIV-1 eradication.
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A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system.
Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
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[Metabolic syndrome: Towards cell therapy by using CRISPR/Cas9-engineered human adipocytes].
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A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing.
Recent studies have reported that genome editing by CRISPR-Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.
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It's a Knockout for Cat Allergies?
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