CRISPR Journal最新文献

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has revolutionized genome editing across various biological systems, including the Apicomplexa phylum. This review describes the status, challenges, and applications of CRISPR-Cas9 editing technology in apicomplexan parasites, such as Plasmodium, Toxoplasma, Theileria, Babesia, and Cryptosporidium. The discussion encompasses successfully implemented CRISPR-Cas9-based techniques in these parasites, highlighting the achieved milestones, from precise gene modifications to genome-wide screening. In addition, the review addresses the challenges hampering efficient genome editing, including the parasites' complex life cycles, multiple intracellular stages, and the lack of robust genetic tools. It further explores the ethical and policy considerations surrounding genome editing and the future perspectives of CRISPR-Cas applications in apicomplexan parasites.

Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene. Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in BRAF^V600E/NRAS^Q61K resistant A375 cells. Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both in vivo and ex vivo gene and cell therapies.

Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.

Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via in vivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to in vivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.

Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34⁺ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.