In vivo genome-wide CRISPR screens identify SOCS1 as intrinsic checkpoint of CD4+ TH1 cell response.

Although CD8+ T cells undergo autonomous clonal proliferation after antigen stimulation in vivo, the expansion of activated CD4+ T cells is limited by intrinsic factors that are poorly characterized. Using genome-wide CRISPR-Cas9 screens and an in vivo system modeling of antigen-experienced CD4+ T cell recruitment and proliferation during a localized immune response, we identified suppressor of cytokine signaling 1 (SOCS1) as a major nonredundant checkpoint imposing a brake on CD4+ T cell proliferation. Using anti­interleukin-2 receptor (IL-2R) blocking antibodies, interferon-γ receptor (IFN-γR) knockout mice, and transcriptomic analysis, we show that SOCS1 is a critical node integrating both IL-2 and IFN-γ signals to block multiple downstream signaling pathways abrogating CD4+ T helper 1 (TH1) cell response. Inactivation of SOCS1 in both murine and human CD4+ T cell antitumor adoptive therapies restored intratumor accumulation, proliferation/survival, persistence, and polyfunctionality and promoted rejection of established tumors. However, in CD8+ T cells, SOCS1 deletion did not affect the proliferation but rather improved survival and effector functions, which allowed for optimal therapeutic outcome when associated with SOCS1 inactivation in CD4+ T cells. Together, these findings identify SOCS1 as a major intracellular negative checkpoint of adoptive T cell response, opening new possibilities to optimize CAR-T cell therapy composition and efficacy.

Programmable Unlocking Nano-Matryoshka-CRISPR Precisely Reverses Immunosuppression to Unleash Cascade Amplified Adaptive Immune Response.

Immune checkpoint blockade (ICB) is an attractive option in cancer therapy, but its efficacy is still less than expected due to the transient and incomplete blocking and the low responsiveness. Herein, an unprecedented programmable unlocking nano-matryoshka-CRISPR system (PUN) targeting programmed cell death ligand 1 (PD-L1) and protein tyrosine phosphatase N2 (PTPN2) is fabricated for permanent and complete and highly responsive immunotherapy. While PUN is inert at normal physiological conditions, enzyme-abundant tumor microenvironment and preternatural intracellular oxidative stress sequentially trigger programmable unlocking of PUN to realize a nano-matryoshka-like release of CRISPR/Cas9. The successful nucleus localization of CRISPR/Cas9 ensures the highly efficient disruption of PD-L1 and PTPN2 to unleash cascade amplified adaptive immune response via revoking the immune checkpoint effect. PD-L1 downregulation in tumor cells not only disrupts PD-1/PD-L1 interaction to attenuate the immunosurveillance evasion but also spurs potent immune T cell responses to enhance adaptive immunity. Synchronously, inhibition of JAK/STAT pathway is relieved by deleting PTPN2, which promotes tumor susceptibility to CD8+ T cells depending on IFN-γ, thus further amplifying adaptive immune responses. Combining these advances together, PUN exhibits optimal antitumor efficiency and long-term immune memory with negligible toxicity, which provides a promising alternative to current ICB therapy.

Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling.

Understanding CRISPR-Cas systems-the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks-is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical 'rich-get-richer' mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.

Production of Human CRISPR-Engineered CAR-T Cells.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy.

The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.

Prophages are associated with extensive CRISPR-Cas auto-immunity.

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales.

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells.

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3-transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3-transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3-transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 transcription, but not protein expression, abolishes CNS2 demethylation. The novel finding that Foxp3-transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.

Synthetic immunomodulation with a CRISPR super-repressor in vivo.

Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors-heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)-are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.

Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.

Immunogold Labeling to Detect Streptococcus pyogenes Cas9 in Cell Culture and Tissues by Electron Microscopy.

The CRISPR-Cas9 system is a powerful and yet precise DNA-editing tool in rapid development. By combining immunogold labeling and electron microscopy with the novel CRISPR-Cas9 system, we propose a new method to gain insight into the biology of this tool. In this study, we analyzed different Cas9-induced systems such as HEK293T cell line, murine oligodendrocyte progenitor cells, brain and liver to detect Cas9 expression by immunoelectron microscopy. Our results show that while Cas9 expression could be found in the nuclei and nucleopores of transfected HEK293T cells, in transfected oligodendrocyte precursor cells, Cas9 was found in cytoplasmic vesicles. In Cas9 constitutively expressing oligodendrocyte precursors, the enzyme was located in the cytoplasm of nondividing cells. Finally, while in the liver Cas9 was detected in different cell types, in the brain we found no specifically labeled cells. In conclusion, immunoelectron microscopy opens a new spectrum of opportunities to study the CRISPR-Cas9 system in a more precise manner.

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence.

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9.

Natural killer cells are potent cytotoxic lymphocytes specialized in recognizing and eliminating transformed cells, and in orchestrating adaptive anti-tumour immunity. However, NK cells are usually functionally exhausted in the tumour microenvironment. Strategies such as checkpoint blockades are under investigation to overcome NK cell exhaustion in order to boost anti-tumour immunity. The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene-editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene-editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells. The current review discusses feasible approaches to deliver the CRISPR/Cas9 systems, as well as potential strategies in gene-editing for NK cell immunotherapy for cancers.

Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci.

CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3-5 that associate with Cas nucleases to specify a viral target for destruction6-9. Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10-12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.