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Clinical trials of germline gene editing: The exploitation problem.
The birth of the world's first genetically edited babies in 2018 provoked considerable ethical outrage. Nonetheless, many scientists and bioethicists now advocate the pursuit of clinical uses of human germline gene editing. Progress towards this goal will require research, including clinical trials where genetically edited embryos are implanted into a woman's uterus, gestated, and brought to term. This paper argues that such trials would likely conflict with the fundamental research ethical requirement of non-exploitation. This is because they would expose people who are in a vulnerable situation to risks and burdens that are substantial and not obviously offset by compensating benefits. I consider how the potential for exploitation in such trials might be mitigated, arguing that a feasible and justifiable approach is not easily found. If this analysis is correct, there is a significant ethical obstacle on the path towards clinical use of human germline gene editing.
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Precise, specific gene editing via a compact GoCas12m-FokI chimeric nuclease.
CRISPR gene editing technologies have transformed functional genomics and biotechnology. Despite these advances, challenges such as limited delivery capacity and off-target activity continue to hinder their therapeutic translation. We developed a chimeric gene editing platform by fusing the compact, catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain. GoCas12m-FokI system integrates the programmable DNA-binding capability of Cas12m with the dimerization-dependent cleavage mechanism of FokI, enabling precise genome editing. Our engineered XTEN-fused GoCas12m-FokI editor exhibits robust activity on both surrogate reporters and endogenous human loci, achieving high-efficiency editing at clinically relevant targets-including CLTA1, HBB, AIFM1, and ABL with no detectable off-target activity at in silico-predicted sites, as confirmed by targeted deep sequencing. Notably, GoCas12m-FokI is nearly half the size of conventional Cas9- or Cas12a-based editors, facilitating delivery via adeno-associated virus and other cargo-limited vectors. This dual-guided editor showed comparable editing efficiency to previously reported FokI-dCas9 systems on endogenous loci, while possessing a different PAM requirement and domain orientation. By combining compact architecture, high specificity, and modular programmability, the GoCas12m-FokI editor offers a powerful alternative for therapeutic genome editing and a promising tool for in vivo gene therapy applications.
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Gene Excision by Dual-Guide CRISPR-Cas9.
CRISPR-Cas9 is frequently used for creating double-strand DNA breaks that result in indels through non-homologous end joining. Indels can revert to wild-type sequence and require sequencing or complex assays to measure. Cutting by two guide RNAs can lead to single indels at either cut site or simultaneous cutting at both sites and repair leading to gene excision.
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A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
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Researchers Use Plasmid Gene Editing to Personalize Solid Tumor T-Cell Therapy.
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Disruption in the DNA Mismatch Repair Gene MSH2 by CRISPR-Cas9 in Indica Rice Can Create Genetic Variability.
Genetic variation is crucial for crop improvement. We adopted a gene editing approach to create variations in the rice genome by targeting the mutator locus homolog 2 (MSH2), a DNA mismatch repair gene. The hypothesis is that disruption of the MSH2 gene leads to a reduced DNA mismatch repair that creates INDELs, resulting in altered phenotypes. The Indica rice (IR-64) genotype was transformed with a guide RNA targeted to the MSH2 gene using an Agrobacterium-mediated in planta method. Many plants showed integration of Cas9 and gRNA constructs in rice plants. One of the msh2 mutants showed a superior phenotype due to editing and possible INDELs in the whole genome. The stable integration of the transgene and its flanking sequence analysis confirms no disruption of any gene, and the observed phenotype is due to the mutations in the MSH2 gene. Few transgenic plants showed disruption of genes due to T-DNA integration that led to altered phenotypes. The plants with altered phenotypes having more tiller number, early flowering, and robust growth with a high biomass were identified. These genetically reprogrammed rice plants could be a potential resource to create more segregating population or act as donor lines to stabilize the important agronomic traits that may help in a speed breeding process.
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A visual monitoring DNA-free multi-gene editing system excised via LoxP::FRT/FLP in poplar.
The CRISPR-Cas9 system has emerged as a potent molecular scalpel for precise genome editing, and profoundly revolutionized plant genetics and breeding, facilitating the development of innovative and improved plant varieties. Typically, the CRISPR-Cas9 gene-editing construct is delivered into target organisms via Agrobacterium tumefaciens-mediated transformation or biolistic methods. However, the incorporation of the CRISPR-Cas9 machinery increases the risk of off-target effects, causing unintended genomic alterations. Additionally, the introduction of exogenous DNA sequences, such as antibiotic resistance marker, raises public concerns regarding the biosafety and regulatory oversight of genetically modified organisms (GMOs), potentially hindering regulatory approval and commercialization. Here, we have engineered an integrated system comprising RUBY, LoxP::FRT/FLP and CRISPR/Cas9-sgRNA cassettes within a single construct, allowing visible color monitoring throughout process including genetic transform, positive transgenic and edited events screening, as well as exogenous DNA excision events, we refer to it as 'a visual monitoring DNA-free multi-gene editing system (VMDFGE)'. This system was introduced into poplar through Agrobacterium tumefaciens-mediated transformation, yielding transgenic poplars with a 75.0% visual screening rate, a 45.8% targeted mutation rate and a 54.5% excision rate for the entire integration system. This approach eliminates the concerns associated with off-target effects and GMO regulatory challenges. It offers significant potential for improvement of poplar, other woody plants and crop species while removing the foreign DNA.
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CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.
Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.
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Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.
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.
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Site-directed RNA editing (SDRE): Off-target effects and their countermeasures.
Site-directed RNA editing (SDRE) is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing. The past few years have witnessed a surge of interest in SDRE, with SDRE tools emerging at a breathtaking pace. However, off-target effects of SDRE remain a tough problem, which constitutes a major hurdle to their clinical applications. Here we discuss the diverse strategies for combating off-target editing, drawing lessons from the published studies as well as our ongoing research. Overall, SDRE is still at its infancy, with significant challenges and exciting opportunities ahead.
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