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The bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) systems, and in particular Streptococcus pyogenes CRISPR–Cas9, have been broadly applied to edit the genome of bacterial and eukaryotic cells. Cas9, which is an RNA-guided programmable nuclease, is a powerful tool for disrupting protein-coding genes. Cas9 cleaves target sites to generate a double-strand break (DSB) that is repaired via an error-prone repair process, leading to insertion/deletion mutations and gene knockouts. However, Cas9 can also be used to modulate genome function without gene disruption, enabling base editing, transcriptional and epigenetic reprogramming, genome imaging, cellular barcoding, genetic recording, and genetic computation. [ABSTRACT FROM AUTHOR]

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has beenshownthatknownNlrp3 stimuli convergeonpotassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wideCRISPR/Cas9screen in immortalizedmousemacrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericininduced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPRassociated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested. [ABSTRACT FROM AUTHOR]

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2′-5′). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2′-5′) is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2′-5′) transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner. [ABSTRACT FROM AUTHOR]

Transcription activator-like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies. [ABSTRACT FROM AUTHOR]

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations. • Tagmentation-based tag integration site sequencing (TTISS) scalably detects DSBs • TTISS is a rapid and streamlined protocol compatible with multiplexing • Application of TTISS highlights trade-off in Cas9 variant specificity and activity • LZ3 Cas9 variant exhibits a unique +1 insertion profile Schmid-Burgk et al. develop tagmentation-based tag integration site sequencing (TTISS), a rapid, streamlined protocol for analyzing double-strand breaks such as those created by CRISPR nucleases. Using TTISS, they comprehensively assess Cas9 variants, revealing a trade-off between specificity and activity and identifying LZ3 Cas9, a variant with a unique +1 insertion profile. [ABSTRACT FROM AUTHOR]

The article reviews the web site BrowserGenome.org which provides RNA sequencing data analysis and visualization.

Cytosolic detection of DNA is crucial for the initiation of antiviral immunity but can also cause autoimmunity in the context of endogenous nucleic acids being sensed. Mutations in the human 3' repair exonuclease 1 (TREX1) have been linked to the type I IFN-associated autoimmune disease Aicardi-Goutières syndrome. The exact mechanisms driving unabated type I IFN responses in the absence of TREX1 are only partly understood, but it appears likely that accumulation of endogenous DNA species triggers a cell-autonomous immune response by activating a cytosolic DNA receptor. In this article, we demonstrate that knocking out the DNA sensor cyclic GMP-AMP synthase completely abrogates spontaneous induction of IFN-stimulated genes in TREX1-deficient cells. These findings indicate a key role of cyclic GMP-AMP synthase for the initiation of self-DNA-induced autoimmune disorders, thus providing important implications for novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses. [ABSTRACT FROM AUTHOR]

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion. [ABSTRACT FROM AUTHOR]

Summary Interleukin-1β (IL-1β) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1β independently of classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an “alternative inflammasome” that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K + efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1β-mediated immune responses and immunopathology in humans. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans -Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKβ activity. [Display omitted] • iPSC-derived human macrophages form an NLRP3 inflammasome independently of NEK7 • NLRP3 priming by IKKβ proceeds independently of transcription and substitutes NEK7 • IKKβ activity constitutes the predominant NLRP3 priming mechanism in human cells • IKKβ drives NEK7-independent priming by recruiting NLRP3 to the phospholipid PI4P The NLRP3 inflammasome causes lytic cell death and inflammation, but its activation mechanism remains enigmatic. Schmacke et al. now show that the kinase IKKβ provides an essential priming signal for inflammasome formation by recruiting NLRP3 to the trans -Golgi network. Human cells predominantly use IKKβ instead of the priming factor NEK7. [ABSTRACT FROM AUTHOR]

Summary The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5′-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5′-triphosphorylated, backbone modifications and the 5′-ppp-linked methylguanosine ( m7 G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5′-terminal nucleotide (N 1 ) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N 1 -2′O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N 1 -2′O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N 1 -2′O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2′O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N 1 -2′O-methylation in RIG-I tolerance of self-RNA. [ABSTRACT FROM AUTHOR]

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 hontolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligandspecific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)l- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1. [ABSTRACT FROM AUTHOR]

Inflammasomes are multiprotein signaling platforms that form upon sensing microbe- or damage-associated molecular patterns. Upon their formation, caspase-1 is activated, leading to the processing of certain proinflammatory cytokines and the initiation of a special type of cell death, known as pyroptosis. Among known inflammasomes, NLRP3 takes on special importance because it appears to be a general sensor of cell stress. Moreover, unlike other inflammasome sensors, NLRP3 inflammasome activity is under additional transcriptional regulation. In this study, we identify the myeloid-specific microRNA miR-223 as another critical regulator of NLRP3 inflammasome activity. miR-223 suppresses NLRP3 expression through a conserved binding site within the 3' untrans-lated region of NLRP3, translating to reduced NLRP3 inflammasome activity. Although miR-223 itself is not regulated by proinflammatory signals, its expression varies among different myeloid cell types. Therefore, given the tight transcriptional control of NLRP3 message itself, miR-223 functions as an important rheostat controlling NLRP3 inflammasome activity. [ABSTRACT FROM AUTHOR]

Summary Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing. [ABSTRACT FROM AUTHOR]