Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 µM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Biodegradable freestanding rare-earth nanosheets promote multimodal imaging and delivers CRISPR-Cas9 plasmid against tumor.

Designing nanomaterials for bio-imaging and drug delivery for advanced cancer therapy with biodegradability and biocompatibility is a promising but challenging frontier. Herein, we assembled biodegradable and biocompatible ultrathin rare-earth erbium/dysprosium nanosheets that improve contrast in multimodal bio-imaging settings (MRI and X-ray CT) and deliver CRISPR-Cas9 plasmid to treat tumors.

AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma.

Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency.

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

Hypoxia-Responsive Gene Editing to Reduce Tumor Thermal Tolerance for Mild-Photothermal Therapy.

Near-infrared (NIR)-light-triggered photothermal therapy (PTT) is usually associated with undesirable damage to healthy organs nearby due to the high temperatures (>50 °C) available for tumor ablation. Low-temperature PTT would therefore have tremendous value for clinical application. Here, we construct a hypoxia-responsive gold nanorods (AuNRs)-based nanocomposite of CRISPR-Cas9 for mild-photothermal therapy via tumor-targeted gene editing. AuNRs are modified with azobenzene-4,4'-dicarboxylic acid (p-AZO) to achieve on-demand release of CRISPR-Cas9 using hypoxia-responsive azo bonds. In the hypoxic tumor microenvironment, the azo groups of the hypoxia-activated CRISPR-Cas9 nanosystem based on gold nanorods (APACPs) are selectively reduced by the overexpression of reductases, leading to the release of Cas9 and subsequent gene editing. Owing to the knockout of HSP90α for reducing the thermal resistance of cancer cells, highly effective tumor ablation both in vitro and in vivo was achieved with APACPs under mild PTT.

A robust CRISPR-Cas12a biosensor coated with metal-organic framework.

Metal-organic frameworks (MOFs) are proposed to protect a CRISPR-associated enzyme/RNA complex from harsh environments, while the complex can be quickly released from MOFs with high efficiency. Therefore, the application of CRISPR-powered biosensing can be more feasible.

Identification of Autophagy-Related Genes as Targets for Senescence Induction Using a Customizable CRISPR-Based Suicide Switch Screen.

Pro-senescence therapies are increasingly being considered for the treatment of cancer. Identifying additional targets to induce senescence in cancer cells could further enable such therapies. However, screening for targets whose suppression induces senescence on a genome-wide scale is challenging, as senescent cells become growth arrested, and senescence-associated features can take 1 to 2 weeks to develop. For a screen with a whole-genome CRISPR library, this would result in billions of undesirable proliferating cells by the time the senescent features emerge in the growth arrested cells. Here, we present a suicide switch system that allows genome-wide CRISPR screening in growth-arrested subpopulations by eliminating the proliferating cells during the screen through activation of a suicide switch in proliferating cells. Using this system, we identify in a genome-scale CRISPR screen several autophagy-related proteins as targets for senescence induction. We show that inhibiting macroautophagy with a small molecule ULK1 inhibitor can induce senescence in cancer cell lines of different origin. Finally, we show that combining ULK1 inhibition with the senolytic drug ABT-263 leads to apoptosis in a panel of cancer cell lines. IMPLICATIONS: Our suicide switch approach allows for genome-scale identification of pro-senescence targets, and can be adapted to simplify other screens depending on the nature of the promoter used to drive the switch.

Small nucleic acids and the path to the clinic for anti-CRISPR.

CRISPR-based therapeutics have entered clinical trials but no methods to inhibit Cas enzymes have been demonstrated in a clinical setting. The ability to inhibit CRISPR-based gene editing or gene targeting drugs should be considered a critical step in establishing safety standards for many CRISPR-Cas therapeutics. Inhibitors can act as a failsafe or as an adjuvant to reduce off-target effects in patients. In this review we discuss the need for clinical inhibition of CRISPR-Cas systems and three existing inhibitor technologies: anti-CRISPR (Acr) proteins, small molecule Cas inhibitors, and small nucleic acid-based CRISPR inhibitors, CRISPR SNuBs. Due to their unique properties and the recent successes of other nucleic acid-based therapeutics, CRISPR SNuBs appear poised for clinical application in the near-term.

Coassembly of nucleus-targeting gold nanoclusters with CRISPR/Cas9 for simultaneous bioimaging and therapeutic genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology enables genome editing with high precision and versatility and has been widely utilized to combat viruses, bacteria, cancers, and genetic diseases. Nonviral nanocarriers can overcome several limitations of viral vehicles, including immunogenicity, inflammation, carcinogenicity, and low versatility, and thus represent promising platforms for CRISPR/Cas9 delivery. Herein, we for the first time develop the application of protamine-capped gold nanoclusters (protamine-AuNCs) as an effective nanocarrier for Cas9-sgRNA plasmid transport and release to achieve efficient genome editing. The protamine-AuNCs integrate the merits of AuNCs and protamine: AuNCs are able to promptly assemble with Cas9-sgRNA plasmids to allow efficient cellular delivery, while the cationic protamine facilitates the effective release of Cas9-sgRNA plasmids into the cellular nucleus. The AuNCs/Cas9-gRNA plasmid nanocomplexes can not only achieve successful gene editing in cells but also knock out the oncogenic gene for cancer therapy. Moreover, the AuNCs with excellent photoluminescence characteristics endow our nanoplatform with the functionality of bioimaging. Overall, our study provides strong evidence that demonstrates protamine-AuNCs as an effective CRISPR/Cas9 delivery tool for gene therapy.

Type I interferon signaling mediates Mycobacterium tuberculosis-induced macrophage death.

Macrophages help defend the host against Mycobacterium tuberculosis (Mtb), the major cause of tuberculosis (TB). Once phagocytized, Mtb resists killing by macrophages, replicates inside them, and leads to their death, releasing Mtb that can infect other cells. We found that the death of Mtb-infected mouse macrophages in vitro does not appear to proceed by a currently known pathway. Through genome-wide CRISPR-Cas9 screening, we identified a critical role for autocrine or paracrine signaling by macrophage-derived type I IFNs in the death of Mtb-infected macrophages in vitro, and blockade of type I IFN signaling augmented the effect of rifampin, a first-line TB drug, in Mtb-infected mice. Further definition of the pathway of type I IFN-mediated macrophage death may allow for host-directed therapy of TB that is more selective than systemic blockade of type I IFN signaling.

Modulating gene regulation to treat genetic disorders.

Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.

Programmable CRISPR-Cas transcriptional activation in bacteria.

Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by more than 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.

Valproic Acid Significantly Improves CRISPR/Cas9-Mediated Gene Editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has emerged as a powerful technology, with the potential to generate transgenic animals. Particularly, efficient and precise genetic editing with CRISPR/Cas9 offers immense prospects in various biotechnological applications. Here, we report that the histone deacetylase inhibitor valproic acid (VPA) significantly increases the efficiency of CRISPR/Cas9-mediated gene editing in mouse embryonic stem cells and embryos. This effect may be caused through globally enhanced chromatin accessibility, as indicate by histone hyperacetylation. Taken together, our results suggest that VPA can be used to increase the efficacy of CRISPR/Cas9 in generating transgenic systems.

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

NK cells mediate clearance of CD8+ T cell-resistant tumors in response to STING agonists.

Several immunotherapy approaches that mobilize CD8+ T cell responses stimulate tumor rejection, and some, such as checkpoint blockade, have been approved for several cancer indications and show impressive increases in patient survival. However, tumors may evade CD8+ T cell recognition via loss of MHC molecules or because they contain few or no neoantigens. Therefore, approaches are needed to combat CD8+ T cell-resistant cancers. STING-activating cyclic dinucleotides (CDNs) are a new class of immune-stimulating agents that elicit impressive CD8+ T cell-mediated tumor rejection in preclinical tumor models and are now being tested in clinical trials. Here, we demonstrate powerful CDN-induced, natural killer (NK) cell-mediated tumor rejection in numerous tumor models, independent of CD8+ T cells. CDNs enhanced NK cell activation, cytotoxicity, and antitumor effects in part by inducing type I interferon (IFN). IFN acted in part directly on NK cells in vivo and in part indirectly via the induction of IL-15 and IL-15 receptors, which were important for CDN-induced NK activation and tumor control. After in vivo administration of CDNs, dendritic cells (DCs) up-regulated IL-15Rα in an IFN-dependent manner. Mice lacking the type I IFN receptor specifically on DCs had reduced NK cell activation and tumor control. Therapeutics that activate NK cells, such as CDNs, checkpoint inhibitors, NK cell engagers, and cytokines, may represent next-generation approaches to cancer immunotherapy.

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems.

Rapid, precise, and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications. Here, we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells. We construct the system by coupling Tet-on 3G and small molecule-assisted shutoff systems, which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules. This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression. The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence, allowing gene function study in a comprehensive expression profile. Our results reveal that the bidirectional control system enables sensitive dosage- and time-dependent regulation for either turn-on or shutoff of gene expression. We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats. The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.

Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

A genome-scale CRISPR/Cas9 knockout screening reveals SH3D21 as a sensitizer for gemcitabine.

Gemcitabine, 2',2'-difluoro-2'-deoxycytidine, is used as a pro-drug in treatment of variety of solid tumour cancers including pancreatic cancer. After intake, gemcitabine is transferred to the cells by the membrane nucleoside transporter proteins. Once inside the cells, it is converted to gemcitabine triphosphate followed by incorporation into DNA chains where it causes inhibition of DNA replication and thereby cell cycle arrest and apoptosis. Currently gemcitabine is the standard drug for treatment of pancreatic cancer and despite its widespread use its effect is moderate. In this study, we performed a genome-scale CRISPR/Cas9 knockout screening on pancreatic cancer cell line Panc1 to explore the genes that are important for gemcitabine efficacy. We found SH3D21 as a novel gemcitabine sensitizer implying it may act as a therapeutic target for improvement of gemcitabine efficacy in treatment of pancreatic cancer.

Cell-Selective Messenger RNA Delivery and CRISPR/Cas9 Genome Editing by Modulating the Interface of Phenylboronic Acid-Derived Lipid Nanoparticles and Cellular Surface Sialic Acid.

Messenger RNA (mRNA) represents an emerging class of nucleic acid therapeutics for genome editing and genetic disease treatment. Delivering exogenous mRNA selectively to cells, however, remains a main challenge to broaden the biomedical application of mRNA and develop targeted gene therapy. Herein, we report cell-selective mRNA delivery and CRISPR/Cas9 genome editing by modulating the interface of phenylboronic acid (PBA) derived lipid nanoparticles (NPs) and cellular surface sialic acid (SA). We design a cationic lipid featuring a PBA group, PBA-BADP, to self-assemble with mRNA into nanoparticles via electrostatic interactions. Importantly, these nanoparticles present free PBA groups on their surface, showing an enhanced cellular uptake by SA-overexpressing cancer cells via the interfacial PBA/SA interaction. It is shown that PBA-BADP/mRNA NPs transfection results in 300 times higher luciferase reporter gene expression in cancer cells than that in noncancer cells. Moreover, we demonstrate that the delivery of tumor suppressor p53 mRNA using PBA-BADP selectively prohibits cancer cell growth, while PBA-BADP/Cas9 mRNA NPs delivery knocks out gene expression of HeLa cancer cells in a much higher efficiency than noncancer cells. We believe these findings could further extend the modulation of PBA and cellular SA interface to advance mRNA delivery and genome editing for new gene therapy.