Despite the clinical success in vascularized composite allotransplantation (VCA), systemic immunosuppression remains necessary to prevent allograft rejection. Even with potent immunosuppressive regimens (tacrolimus, mycophenolate mofetil, and steroids), most patients experience several rejection episodes, often within the same year. The risk of systemic side effects must constantly be weighed against the risk of under-immunosuppression and, thus, acute and chronic rejection. In this context, genomic editing has emerged as a potential tool to minimize the need for toxic immunosuppressive regimens and has gained attention in the fields of solid organ transplantation and xenotransplantation. This strategy may also be relevant for the future of VCA.
We discuss the topic of genetic engineering and review recent developments in this field that justify investigating tools such as clustered regularly interspaced short palindromic repeats/Cas9 in the context of VCA.
We propose specific strategies for VCA based on the most recent gene expression data. This includes the well-known strategy of tolerance induction. Specifically, targeting the interaction between antigen-presenting cells and recipient-derived T cells by CD40 knockout may be effective. The novelty for VCA is a discovery that donor-derived T lymphocytes may play a special role in allograft rejection of facial transplants. We suggest targeting these cells prior to transplantation (e.g., by ex vivo perfusion of the transplant) by knocking out genes necessary for the long-term persistence of donor-derived immune cells in the allograft.
Despite the demonstrated feasibility of VCA in recent years, continued improvements to immunomodulatory strategies using tools like clustered regularly interspaced short palindromic repeats/Cas9 could lead to the development of approaches that mitigate the limitations associated with rejection of this life-giving procedure.

The clinical outcome for children and adolescents with homozygous familial hypercholesterolaemia (HoFH) can be devastating, and treatment options are limited in the presence of a null variant. In HoFH, atherosclerotic risk accumulates from birth. Gene therapy is an appealing treatment option as restoration of low-density lipoprotein receptor (LDLR) gene function could provide a cure for HoFH. A clinical trial using a recombinant adeno-associated vector (rAAV) to deliver LDLR DNA to adult patients with HoFH was recently completed; results have not yet been reported. However, this treatment strategy may face challenges when translating to the paediatric population. The paediatric liver undergoes substantial growth which is significant as rAAV vector DNA persists primarily as episomes (extra-chromosomal DNA) and are not replicated during cell division. Therefore, rAAV-based gene addition treatment administered in childhood would likely only have a transient effect. With over 2,000 unique variants in LDLR, a goal of genomic editing-based therapy development would be to treat most (if not all) mutations with a single set of reagents. For a robust, durable effect, LDLR must be repaired in the genome of hepatocytes, which could be achieved using genomic editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and a DNA repair strategy such as homology-independent targeted integration. This review discusses this issue in the context of the paediatric patient group with severe compound heterozygous or homozygous null variants which are associated with aggressive early-onset atherosclerosis and myocardial infarction, together with the important pre-clinical studies that use genomic editing strategies to treat HoFH in place of apheresis and liver transplantation.

Effective and long-term treatment is required for controlling chronic Hepatitis B Virus (HBV) infection. Natural killer (NK) cells are antiviral innate lymphocytes and represent an essential arm of current immunotherapy. In chronic HBV (CHB), NK cells display altered changes in phenotypes and functions, but preserve antiviral activity, especially for cytolytic activity. On the other hand, NK cells might also cause liver injury in the disease. NK -based immunotherapy, including adoptive NK cell therapy and NK -based checkpoint inhibition, could potentially exploit the antiviral aspect of NK cells for controlling CHB infection while preventing liver tissue damage. Here, we review recent progress in NK cell biology under the context of CHB infection, and discuss potential NK -based immunotherapy strategies for the disease.

Human genome editing has been increasingly explored to determine if it can be used to eradicate genetic diseases like sickle cell disease, but it has also been surrounded by a wide variety of ethical dilemmas. The purpose of this review was to conduct a scoping review of the ethics of therapeutic human genome editing in terms of philosophy, theology, public perspectives, and research ethics. A systemized search of PubMed, Embase, Ovid MEDLINE, and Web of Science was conducted. The initial search resulted in 4,445 articles, and after removing 1,750 duplicates and screening the remaining 2,695 articles, 27 final articles were selected for the final analysis. From a philosophical and theological standpoint, therapeutic human genome editing was generally ethically acceptable. Worldwide public perspectives were also in agreement except for the Oceanic region, which disagreed mainly due to the possible effects on future generations. Lastly, human research ethics revealed that women were not always included in informed consent, and that child autonomy needs to be preserved. Further research is needed to determine adverse effects on the mother, fetus, and future generations.

CRISPR/Cas9 systems were established in some edible fungi based on in vivo expressed Cas9 and guide RNA. Compared with those systems, the in vitro assembled Cas9 and sgRNA ribonucleoprotein complexes (RNPs) have more advantages, but only a few examples were reported, and the editing efficiency is relatively low. In this study, we developed and optimized a CRISPR/Cas9 genome-editing method based on in vitro assembled ribonucleoprotein complexes in the mushroom Flammulina filiformis. The surfactant Triton X-100 played a critical role in the optimal method, and the targeting efficiency of the genomic editing reached 100% on a selective medium containing 5-FOA. This study is the first to use an RNP complex delivery to establish a CRISPR/Cas9 genome-editing system in F. filiformis. Moreover, compared with other methods, this method avoids the use of any foreign DNA, thus saving time and labor when it comes to plasmid construction.

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the \"knocking out\" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.

The history of DNA manipulation for the creation of genetically modified animals began in the 1970s, using viruses as the first DNA molecules microinjected into mouse embryos at different preimplantation stages. Subsequently, simple DNA plasmids were used to microinject into the pronuclei of fertilized mouse oocytes and that method became the reference for many years. The isolation of embryonic stem cells together with advances in genetics allowed the generation of gene-specific knockout mice, later on improved with conditional mutations. Cloning procedures expanded the gene inactivation to livestock and other non-model mammalian species. Lentiviruses, artificial chromosomes, and intracytoplasmic sperm injections expanded the toolbox for DNA manipulation. The last chapter of this short but intense history belongs to programmable nucleases, particularly CRISPR-Cas systems, triggering the development of genomic-editing techniques, the current revolution we are living in.

Introduction Breast cancer is the leading cause of cancer-related deaths in women worldwide with the majority of deaths due to metastasis. The development of metastasis is closely related to the tumor microenvironment where tumor-associated macrophages (TAMs) are the main immune cell component playing a crucial role in tumor migration. Key players in tumor progression, metastasis and survival are the receptor CXCR4 and its ligand CXCL12. CXCR4 is expressed in multiple cell types including macrophages and breast cancer cells. Many studies have focus on the role of CXCR4 expressed in breast cancer cells. Methods In this study, we investigated the role of CXCR4 expressed in TAMs on breast cancer cell migration by reducing CXCR4 expression via CRISPR-CAS9 system in differentiated THP-1 cells (a TAMs model). Results According to wound healing migration assay, MCF7 cancer cells co-cultured with genetically edited dTHP-1 cells have a lower migration rate as compared to MCF7 cancer cells co-cultured with unedited and dTHP-1 cells. Conclusion The study demonstrates the role of CXCR4 on breast cancer cell migration through TAM-cancer cell crosstalk.

Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.

The potential of the cluster regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9)-based therapeutic genome editing is severely hampered by the difficulties in precise regulation of the in vivo activity of the CRISPR-Cas9 system. Herein, sono-controllable and reactive oxygen species (ROS)-sensitive sonosensitizer-integrated metal-organic frameworks (MOFs), denoted as P/M@CasMTH1, are developed for augmented sonodynamic therapy (SDT) efficacy using the genome-editing technology. P/M@CasMTH1 nanoparticles comprise singlet oxygen (1 O2 )-generating MOF structures anchored with CRISPR-Cas9 systems via 1 O2 -cleavable linkers, which serve not only as a delivery vector of CRISPR-Cas9 targeting MTH1, but also as a sonoregulator to spatiotemporally activate the genome editing. P/M@CasMTH1 escapes from the lysosomes, harvests the ultrasound (US) energy and converts it into abundant 1 O2 to induce SDT. The generated ROS subsequently trigger cleavage of ROS-responsive thioether bonds, thus inducing controllable release of the CRISPR-Cas9 system and initiation of genome editing. The genomic disruption of MTH1 conspicuously augments the therapeutic efficacy of SDT by destroying the self-defense system in tumor cells, thereby causing cellular apoptosis and tumor suppression. This therapeutic strategy for synergistic MTH1 disruption and abundant 1 O2 generation provides a paradigm for augmenting SDT efficacy based on the emerging nanomedicine-enabled genome-editing technology.