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The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage. A new DNA ‘base editor’ can change targeted A•T base pairs to G•C, allowing disease-associated mutations to be corrected and disease-suppressing mutations to be introduced into cells. Base editing steps forward In 2016, David Liu and colleagues developed a DNA 'base editor'—a system that would make it possible to change C•G base pairs to T•A base pairs within DNA without introducing double-stranded breaks. This approach involves tethering of a cytidine deaminase to an inactive RNA-guided Cas9 complex that enables site selectivity. However, this system was unable to correct about half of the single nucleotide polymorphisms that are known to be pathogenic. Now, David Liu and collaborators describe the next step in genomic base editing technology, designed to tackle the conversion of A•T base pairs to G•C base pairs. Beginning with a bacterial adenosine deaminase that acts on RNA, they used seven rounds of selection and refinement to produce ABE7.10. This enzyme, again tethered to an inactive RNA-guided Cas9 complex, uses DNA as a substrate and resulted in an average correction efficiency of 53% across multiple sites and contexts in the genome, with a very low mutagenic background. Importantly, the system can be used both to correct disease-associated single nucleotide polymorphisms and to introduce disease-suppressing ones.

The cGAS–STING signalling axis, comprising the synthase for the second messenger cyclic GMP–AMP (cGAS) and the cyclic GMP–AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS–STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS–STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid–liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS–STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved.The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway senses DNA in the cytoplasm, whether of pathogenic or endogenous (chromatin or mitochondrial) origin, and triggers the interferon response. The mechanisms of DNA recognition and cGAS–STING activation and signalling are now coming into focus, providing insights into the cellular functions of this pathway, including interferon-independent roles.

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage 1 – 4 , and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood 5 – 7 . Although Cas9 variants with greater discrimination against mismatches have been designed 8 – 10 , these suffer from substantially reduced rates of on-target DNA cleavage 5 , 11 . Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants. Cryo-electron microscopy structures of Cas9 during mismatch cleavage provide insight into the mechanisms that control off-target effects of Cas9, which will aid in the future design of high-fidelity Cas9 variants with reduced off-target cleavage.

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species 1 , 2 . However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response. By developing a new eccDNA purification and profiling method, the study revealed close-to-random genomic origination, mechanism of biogenesis and function of eccDNAs. 

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.Deficiency in the protein kinase ATM — a master regulator of double-strand DNA breaks and stress responses — causes ataxia telangiectasia (A-T). Recent studies link A-T with other neurodegenerative disorders, and implicate reactive oxygen species, mitochondrial dysfunction, defects in proteostasis and metabolism, and increased poly(ADP-ribosyl)ation in the aetiology of A-T.

In pathophysiology, reactive oxygen species control diverse cellular phenotypes by oxidizing biomolecules. Among these, the guanine base in nucleic acids is the most vulnerable to producing 8-oxoguanine, which can pair with adenine. Because of this feature, 8-oxoguanine in DNA (8-oxo-dG) induces a G > T (C > A) mutation in cancers, which can be deleterious and thus actively repaired by DNA repair pathways. 8-Oxoguanine in RNA (o 8 G) causes problems in aberrant quality and translational fidelity, thereby it is subjected to the RNA decay pathway. In addition to oxidative damage, 8-oxo-dG serves as an epigenetic modification that affects transcriptional regulatory elements and other epigenetic modifications. With the ability of o 8 G•A in base pairing, o 8 G alters structural and functional RNA–RNA interactions, enabling redirection of posttranscriptional regulation. Here, we address the production, regulation, and function of 8-oxo-dG and o 8 G under oxidative stress. Primarily, we focus on the epigenetic and epitranscriptional roles of 8-oxoguanine, which highlights the significance of oxidative modification in redox-mediated control of gene expression. Genetics: modification of guanine base has far-reaching consequences Emerging evidence suggests the modified base 8-oxoguanine, created when damaging chemicals called reactive oxygen species interact with DNA and RNA, has more extensive effects on cell function than previously suspected. This modified form of guanine, one of the key chemical ‘bases’ of DNA and RNA can cause mutations in DNA and disrupt the functioning of the RNA copies of DNA that mediate gene expression. These problems occur because 8-oxoguanine can aberrantly pair up with the base adenine in crucial DNA and RNA interactions, generating mutations that can cause cancers and other conditions. Sung Wook Chi and colleagues at Korea University in Seoul, review research offering evidence of the wider consequences of 8-oxoguanine formation, especially disruption of the complex processes that regulate the activity of genes and RNA. Further investigation of the medical implications is warranted.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division. Diehl et al. show that imbalance among nucleotide species is not sensed by canonical metabolic regulatory pathways, causing excessive cell growth despite a DNA replication block. ATR is needed to increase nucleotide availability in normal S phase.

Replication protein A (RPA) and RAD51 are DNA-binding proteins that help maintain genome stability during DNA replication. These proteins regulate nucleases, helicases, DNA translocases, and signaling proteins to control replication, repair, recombination, and the DNA damage response. Their different DNA-binding mechanisms, enzymatic activities, and binding partners provide unique functionalities that cooperate to ensure that the appropriate activities are deployed at the right time to overcome replication challenges. Here we review and discuss the latest discoveries of the mechanisms by which these proteins work to preserve genome stability, with a focus on their actions in fork reversal and fork protection.

Crystal structure of the RNA-guided endonuclease Cas9 bound to a guide RNA and a target DNA duplex reveals how base-specific recognition of a short motif known as PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA. PAM-mediated DNA target opening The bacterial Cas9 nuclease is an RNA-guided DNA endonuclease found in bacterial CRISPR defence systems and is also widely used as a genetic engineering tool. Cas9 associates with a guide RNA, forms a complex with complementary duplex DNA, and cleaves the DNA. For cleavage to occur, the DNA target must contain a trinucleotide motif known as PAM. Martin Jinek and colleagues have solved the structure of Cas9 bound to a guide RNA and a PAM-containing duplex DNA. The structure reveals how base-specific recognition of PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA. The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA 1 , 2 . Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control 3 . RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA 1 , 4 , 5 , 6 . Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5′-NGG-3′ PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA–DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends 1 , 2 . BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1) 3 – 8 . When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors 4 , 6 – 9 . Here we address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. We report that CTC1–STN1–TEN1 (CST) 10 , a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase 11 , is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin. 53BP1 and shieldin recruit the CTC1–STN1–TEN1 complex and polymerase-α to sites of DNA damage to help control the repair of double-strand breaks.