CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O$_{2}$-dependent shuttle of oxidizable fatty acid substrates. MB’s impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively

Antiviral signaling by a cyclic nucleotide activated CRISPR protease

Funding information: M.G. and J.L.S.B. are funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC2151–390873048. M.F.W. acknowledges a European Research Council Advanced Grant (grant number 101018608) and the China Scholarship Council (REF: 202008420207 to H.C.). G.H. is grateful for funding by the Deutsche Forschungsgemeinschaft (grant number HA6805/6-1).CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition3-5. Among a large set of proteins that were linked to type III systems and predicted to bind cOAs6,7, a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODS-associated and fused to various effector domains) family7, fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazF-homolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS).PostprintPeer reviewe

TREX1 Deficiency Triggers Cell-Autonomous Immunity in a cGAS-Dependent Manner

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

RNA-guided DNA insertion with CRISPR-associated transposases

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.National Institutes of Health (Grant 1R01-HG009761)National Institutes of Health (Grant 1R01-MH110049)National Institutes of Health (Grant 1DP1-HL141201

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP

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

Diverse enzymatic activities mediate antiviral immunity in prokaryotes

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