Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing.

CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites.

CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

Pd Anchored on a Phytic Acid/Thiourea Polymer as a Highly Active and Stable Catalyst for the Reduction of Nitroarene.

A kind of N, P, C, O-containing polymer was easily prepared via microwave heating of phytic acid and thiourea just for 90 s. After impregnation and reduction of H2PdCl4, highly dispersed Pd single atoms/sub-nano clusters loaded on the phytic acid/thiourea polymer (Pd-CNSP) were successfully obtained. Owing to the synergetic effect of the polymer support and Pd, the catalyst Pd-CNSP achieves a great atomic efficiency of Pd species and exhibits an outstanding catalytic ability in the reduction of 4-nitrophenol. The k value of the catalyst Pd-CNSP (2.17 min-1 mg-1) is about 19 times higher than that of the commercial Pd/C (5 wt %) catalyst. The turnover frequency value is as high as 848 min-1, which is the highest value reported so far. Pd-CNSP also has good selectivity for the reduction of halogen-substituted (Cl and Br) nitroaromatics. It is expected to be mass-produced and used in other industrial hydrogenation reactions.

Novel Mechanism for Cyclic Dinucleotide Degradation Revealed by Structural Studies of Vibrio Phosphodiesterase V-cGAP3.

3'3'-cyclic GMP-AMP (3'3'-cGAMP) belongs to a family of the bacterial secondary messenger cyclic dinucleotides. It was first discovered in the Vibrio cholerae seventh pandemic strains and is involved in efficient intestinal colonization and chemotaxis regulation. Phosphodiesterases (PDEs) that degrade 3'3'-cGAMP play important regulatory roles in the relevant signaling pathways, and a previous study has identified three PDEs in V. cholerae, namely, V-cGAP1, V-cGAP2, and V-cGAP3, functioning in 3'3'-cGAMP degradation. We report the crystal structure, biochemical, and structural analyses of V-cGAP3, providing a foundation for understanding the mechanism of 3'3'-cGAMP degradation and regulation in general. Our crystal and molecular dynamic (MD)-simulated structures revealed that V-cGAP3 contains tandem HD-GYP domains within its N- and C-terminal domains, with similar three-dimensional topologies despite their low-sequence identity. Biochemical and structural analyses showed that the N-terminal domain plays a mechanism of positive regulation for the catalytic C-terminal domain. We also demonstrated that the other homologous Vibrio PDEs, V-cGAP1/2, likely function via a similar mechanism.

MAVS activates TBK1 and IKKε through TRAFs in NEMO dependent and independent manner.

Mitochondrial antiviral-signaling protein (MAVS) transmits signals from RIG-I-like receptors after RNA virus infections. However, the mechanism by which MAVS activates downstream components, such as TBK1 and IKKα/ß, is unclear, although previous work suggests the involvement of NEMO or TBK1-binding proteins TANK, NAP1, and SINTBAD. Here, we report that MAVS-mediated innate immune activation is dependent on TRAFs, partially on NEMO, but not on TBK1-binding proteins. MAVS recruited TBK1/IKKε by TRAFs that were pre-associated with TBK1/IKKε via direct interaction between the coiled-coil domain of TRAFs and the SDD domain of TBK1/IKKε. TRAF2-/-3-/-5-/-6-/- cells completely lost RNA virus responses. TRAFs' E3 ligase activity was required for NEMO activation by synthesizing ubiquitin chains that bound to NEMO for NF-κB and TBK1/IKKε activation. NEMO-activated IKKα/ß were important for TBK1/IKKε activation through IKKα/ß-mediated TBK1/IKKε phosphorylation. Moreover, individual TRAFs differently mediated TBK1/IKKε activation and thus fine-tuned antiviral immunity under physiological conditions.

Nonspecific DNA Binding of cGAS N Terminus Promotes cGAS Activation.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediates innate immune responses against invading pathogens, or against self-dsDNA, which causes autoimmune disorders. Upon nonspecific binding of cytosolic B-form DNA, cGAS synthesizes the second messenger 2'3'-cGAMP and triggers STING-dependent signaling to produce type I IFNs. The cGAS comprises less-conserved N-terminal residues and highly conserved nucleotidyltransferase/Mab21 domains. The function and structure of the well-conserved domains have been extensively studied, whereas the physiological function of the N-terminal domain of cGAS is largely uncharacterized. In this study we used a single-molecule technique combined with traditional biochemical and cellular assays to demonstrate that binding of nonspecific dsDNA by the N-terminal domain of cGAS promotes its activation. We have observed that the N terminus of human cGAS (hcGAS-N160) undergoes secondary structural change upon dsDNA binding in solution. Furthermore, we showed that the hcGAS-N160 helps full length hcGAS to expand the binding range on λDNA and facilitates its binding efficiency to dsDNA compared with hcGAS without the 160 N-terminal residues (hcGAS-d160). More importantly, hcGAS-N160 endows full length hcGAS relatively higher enzyme activity and stronger activation of STING/IRF3-mediated cytosolic DNA signaling. These findings strongly indicate that the N-terminal domain of cGAS plays an important role in enhancing its function.

cGAS-cGAMP-STING: The three musketeers of cytosolic DNA sensing and signaling.

Innate immunity is the first line of host defense against invading pathogens. The detection of aberrant nucleic acids which represent some conserved PAMPs triggers robust type I IFN-mediated innate immune responses. Host- or pathogen-derived cytosolic DNA binds and activates the DNA sensor cGAS, which synthesizes the second messenger 2'3'-cGAMP and triggers STING-dependent downstream signaling. Here, we highlight recent progress in cGAS-cGAMP-STING, the Three Musketeers of cytosolic DNA sensing and signaling, and their essential roles in infection, autoimmune diseases, and cancer. We also focus on the regulation of these critical signal components by variant host/pathogen proteins and update our understanding of this indispensable pathway to provide new insights for drug discovery. © 2016 IUBMB Life, 68(11):858-870, 2016.

Cyclic (di)nucleotides: the common language shared by microbe and host.

Fluency in a common language allows individuals to convey information and carry out complex activities that otherwise would be difficult or even impossible without the benefit of shared communication. Cyclic (di)nucleotides have recently been recognized as such an accessible language understood by both microbe and the host, ever since remarkable progresses have revealed the molecular details of these nucleotide second messengers used in cellular communication systems. Though undergoing separate evolutionary pathways in prokaryotes and eukaryotes, cyclic (di)nucleotides enable microbes to influence host cells immediately and fiercely by modulating a variety of cellular activities. Here we highlight recent insights in cyclic (di)nucleotides and focus on the balancing of these indispensable signaling molecules by synthases and phosphodiesterases.

Rat and human STINGs profile similarly towards anticancer/antiviral compounds.

Cyclic dinucleotides (CDNs) and antitumor/antiviral agents (DMXAA and CMA) trigger STING-dependent innate immunity activation. Accumulative evidences have showed that DMXAA and CMA selectively activate mouse, but not human STING signaling. The mechanism underlying this species selectivity remains poorly understood. In this report, we have shown that human and rat STINGs display more similar signaling profiles toward DMXAA and CMA than that of human and mouse STINGs, suggesting that rat is more suitable for preclinical testing of STING-targeted drugs. We have also determined the crystal structures of both apo rat STING and its complex with cyclic GMP-AMP with 2'5' and 3'5' phosphodiester linkage (2'3'-cGAMP), a human endogenous CDN. Structure-guided biochemical analysis also revealed the functional importance of the connecting loop (A140-N152) between membrane and cytosolic domains in STING activation. Taken together, these findings reveal that rat STING is more closely related to human STING in terms of substrate preference, serving as a foundation for the development of STING-targeted drugs.

Identification and characterization of phosphodiesterases that specifically degrade 3'3'-cyclic GMP-AMP.

Cyclic dinucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Although phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP-specific PDEs in V. cholerae (herein designated as V-cGAP1/2/3). V-cGAPs are HD-GYP domain-containing proteins and specifically break 3'3'-cGAMP, but not other forms of cGAMP. 3'3'-cGAMP is first linearized by all three V-cGAPs to produce 5'-pApG, which is further hydrolyzed into 5'-ApG by V-cGAP1. In this two-step reaction, V-cGAP1 functions as both a PDE and a 5'-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3'3'-cGAMP suggests the existence of specific PDEs for other cGAMPs, including 2'3'-cGAMP in mammalian cells. The absolute requirement of the GYP motif for 3'3'-cGAMP degradation suggests that HD domain-containing PDEs in eukaryotes are probably unable to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3'3'-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize a similar strategy. These results will provide valuable information for identification and characterization of mammalian 2'3'-cGAMP-specific PDEs in future studies.