Explore the vast range of titles available.

The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.

Immunotherapy of cancer with CD3-bispecific antibodies is an approved therapeutic option for some hematological malignancies and is under clinical investigation for solid cancers. However, the treatment of solid tumors faces more pronounced hurdles, such as increased on-target off-tumor toxicities, sparse T-cell infiltration and impaired T-cell quality due to the presence of an immunosuppressive tumor microenvironment, which affect the safety and limit efficacy of CD3-bispecific antibody therapy. In this review, we provide a brief status update of the CD3-bispecific antibody therapy field and identify intrinsic hurdles in solid cancers. Furthermore, we describe potential combinatorial approaches to overcome these challenges in order to generate selective and more effective responses.

How single-chain variable fragments (scFvs) affect the functions of chimeric antigen receptors (CARs) has not been well studied. Here, the components of CAR with an emphasis on scFv were described, and then several methods to measure scFv affinity were discussed. Next, scFv optimization studies for CD19, CD38, HER2, GD2 or EGFR were overviewed, showing that tuning the affinity of scFv could alleviate the on-target/off-tumor toxicity. The affinities of scFvs for different antigens were also summarized to designate a relatively optimal working range for CAR design. Last, a synthetic biology approach utilizing a low-affinity synthetic Notch (synNotch) receptor to achieve ultrasensitivity of antigen-density discrimination and murine models to assay the on-target/off-tumor toxicity of CARs were highlighted. Thus, this review provides preliminary guidelines of choosing the right scFvs for CARs.

The target of rapamycin (TOR) kinase, a master regulator that is evolutionarily conserved among yeasts (Saccharomyces cerevisiae), plants, animals, and humans, integrates nutrient and energy signaling to promote cell proliferation and growth. Recent breakthroughs made possible by integrating chemical, genetic, and genomic analyses have greatly increased our understanding of the molecular functions and dynamic regulation of the TOR kinase in photosynthetic plants. TOR signaling plays fundamental roles in embryogenesis, meristem activation, root and leaf growth, flowering, senescence, and life span determination. The molecular mechanisms underlying TOR-mediated ribosomal biogenesis, translation promotion, readjustment of metabolism, and autophagy inhibition are now being uncovered. Moreover, monitoring photosynthesis-derived GѪc and bioenergetics relays has revealed that TOR orchestrates unprecedented transcriptional networks that wire central metabolism and biosynthesis for energy and biomass production. In addition, these networks integrate localized stem/progenitor cell proliferation through interorgan nutrient coordination to control developmental transitions and growth.

The approval of two chimeric antigen receptor-modified T cell types by the US Food and Drug Administration (FDA) for the treatment of hematologic malignancies is a milestone in immunotherapy; however, the application of CAR-T cells has been limited by antigen escape and on-target, off-tumor toxicities. Therefore, it may be a potentially effective strategy to select appropriate targets and to combine multi-antigen-targeted CAR-T cells with “OR”, “AND” and “NOT” Boolean logic gates. We summarize the current limitations of CAR-T cells as well as the efficacy and safety of logic-gated CAR-T cells in antitumor therapy. This review will help to explore more optimized strategies to expand the CAR-T cell therapeutic window.

Background Genome-wide knockout studies, noncoding deletion scans, and other large-scale studies require a simple and lightweight framework that can quickly discover and score thousands of candidate CRISPR guides targeting an arbitrary DNA sequence. While several CRISPR web applications exist, there is a need for a high-throughput tool to rapidly discover and process hundreds of thousands of CRISPR targets. Results Here, we introduce FlashFry, a fast and flexible command-line tool for characterizing large numbers of CRISPR target sequences. With FlashFry, users can specify an unconstrained number of mismatches to putative off-targets, richly annotate discovered sites, and tag potential guides with commonly used on-target and off-target scoring metrics. FlashFry runs at speeds comparable to commonly used genome-wide sequence aligners, and output is provided as an easy-to-manipulate text file. Conclusions FlashFry is a fast and convenient command-line tool to discover and score CRISPR targets within large DNA sequences.

Chimeric antigen receptor (CAR)‐T cell therapy is a transformative treatment against advanced malignancies. Unfortunately, once administrated in vivo, CAR‐T cells become out of artificial control, and fierce response to CAR‐T therapy may cause severe adverse events, represented by cytokine‐release syndrome and on‐target/off‐tumor effects. Here, a nanomodified switch strategy is developed, leading to sustained and precise “on‐tumor only” activation of CAR‐T cells. Here, original gelatinase‐responsive nanoparticles (NPs) are used to selectively deliver the heterodimerizing switch, which is the key component of switchable CAR with separated activation modules. The “NanoSwitch” is tumor‐specific, thus inactivated switchable CAR‐T cells do little harm to normal cells, even if the normal cells express the target of CAR‐T. Owing to the sustained‐release effect of NPs, the CAR‐T cells are activated smoothly, avoiding sudden release of cytokine. These data introduce NanoSwitch as a universal and applicable solution to safety problems of CAR‐T therapy regardless of the target. Here, nanoparticles based on gelatinase‐responsive strategy are used to construct “NanoSwitch” to control the activation process of chimeric antigen receptor (CAR)‐T cells, leading to on‐site activation of CAR‐T cells at the tumor region in a precise and sustainable way. This platform has promising potential to prevent the main obstacles in the CAR‐T therapy for solid tumors including cytokine‐release syndrome and on‐target/off‐tumor toxicity.

The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ∼3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites,which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes.

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Antibody‐based therapies could be led astray when target receptors are expressed on nontarget sites, and the on‐target toxicity poses critical challenges to clinical applications. Here, a biomimetic indirect active targeting (INTACT) strategy is proposed based on receptor expression disparities between nontarget sites and the targets. By prebinding the antibodies using cell membrane vesicles with appropriate receptor expressions, the INTACT strategy could filter out the interactions on nontarget sites due to their inferior receptor expression, whereas ensure on‐demand release at the targets by competitive binding. The strategy is verified on CD47 antibody, realizing drastic alleviation of its clinically concerned hematotoxicity on a series of animal models including humanized patient‐derived xenograft platforms, accompanied by preferable therapeutic effects. Furthermore, the INTACT strategy proves extensive applicability for various systems including antibody, antibody–drug conjugate, and targeted delivery systems, providing a potential platform refining the specificity for frontier antibody‐related therapies. By prebinding the antibodies using cell membrane vesicles with appropriate receptor expressions, the indirect active targeting (INTACT) strategy could filter out the interactions on nontarget sites due to inferior receptor expression while ensuring on‐demand release at the targets by competitive binding. This study provides a potential platform for refining the specificity for frontier antibody‐related therapies.