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Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks 1 – 3 , but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA 4 – 8 , it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA 9 – 13 . For example, the cytosine deaminase APOBEC1—which is used in cytosine base editors (CBEs)—targets both DNA and RNA 12 , and the adenine deaminase TadA—which is used in adenine base editors (ABEs)—induces site-specific inosine formation on RNA 9 , 11 . However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern 14 – 16 . Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases. Cytosine and adenine base editors have undesired off-target effects on RNA, but this activity can be reduced in deaminase-engineered variants while preserving on-target DNA editing.

A dose-finding study showed that CMX001 at an oral dose of 100 mg twice weekly, as compared with placebo, reduced the risk of cytomegalovirus events from 37% to 10% among patients who had undergone hematopoietic-cell transplantation. Cytomegalovirus (CMV) infection is a common cause of illness after allogeneic hematopoietic-cell transplantation. 1 , 2 CMV seropositivity in transplant recipients is also associated with an increased risk of death after transplantation, despite preemptive and prophylactic strategies with available antiviral agents. 3 – 5 Although valganciclovir is approved for prophylaxis against CMV infection after solid-organ transplantation, its use is limited by myelosuppression, particularly after hematopoietic-cell transplantation. 3 , 6 – 8 Thus, there is an unmet need for effective drugs against CMV infection that have a better safety profile. CMX001 is an orally bioavailable lipid acyclic nucleoside phosphonate that is absorbed in the small intestine and transported . . .

The nucleotide analogue brincidofovir was developed to prevent and treat infections caused by double-stranded DNA viruses. Based on in vitro data suggesting an antiviral effect against Ebola virus, brincidofovir was included in the World Health Organisation list of agents that should be prioritised for clinical evaluation in patients with Ebola virus disease (EVD) during the West African epidemic. In this single-arm phase 2 trial conducted in Liberia, patients with laboratory-confirmed EVD (two months of age or older, enrolment bodyweight ≥50 kg) received oral brincidofovir 200 mg as a loading dose on day 0, followed by 100 mg brincidofovir on days 3, 7, 10, and 14. Bodyweight-adjusted dosing was used for patients weighing <50 kg at enrolment. The primary outcome was survival at Day 14 after the first dose of brincidofovir. Four patients were enrolled between 01 January 2015 and 31 January 2015. The trial was stopped following the decision by the manufacturer to terminate their program of development of brincidofovir for EVD. No Serious Adverse Reactions or Suspected Unexpected Serious Adverse Reactions were identified. All enrolled subjects died of an illness consistent with EVD. Due to the small sample size it was not possible to determine the efficacy of brincidofovir for the treatment of EVD. The premature termination of the trial highlights the need to establish better practices for preclinical in-vitro and animal screening of therapeutics for potentially emerging epidemic infectious diseases prior to their use in patients. Pan African Clinical Trials Registry PACTR201411000939962.

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.

Vulval intraepithelial neoplasia is a skin disorder affecting the vulva that, if left untreated, can become cancerous. Currently, the standard treatment for patients with vulval intraepithelial neoplasia is surgery, but this approach does not guarantee cure and can be disfiguring, causing physical and psychological problems, particularly in women of reproductive age. We aimed to assess the activity, safety, and feasibility of two topical treatments—cidofovir and imiquimod—as an alternative to surgery in female patients with vulval intraepithelial neoplasia. We recruited female patients (age 16 years or older) from 32 centres to an open-label, randomised, phase 2 trial. Eligibility criteria were biopsy-proven vulval intraepithelial neoplasia grade 3 and at least one lesion that could be measured accurately. We randomly allocated patients to topical treatment with either 1% cidofovir (supplied as a gel in a 10 g tube, to last 6 weeks) or 5% imiquimod (one 250 mg sachet for every application), to be self-applied three times a week for a maximum of 24 weeks. Randomisation (1:1) was done by stratified minimisation via a central computerised system, with stratification by hospital, disease focality, and presentation stage. The primary endpoint was a histologically confirmed complete response at the post-treatment assessment visit 6 weeks after the end of treatment (a maximum of 30 weeks after treatment started). Analysis of the primary endpoint was by intention to treat. Secondary outcomes were toxic effects (to assess safety) and adherence to treatment (to assess feasibility). We present results after all patients had reached the primary endpoint assessment point at 6 weeks; 2-year follow-up of complete responders continues. This trial is registered with Current Controlled Trials, ISRCTN 34420460. Between Oct 21, 2009, and Jan 11, 2013, 180 participants were enrolled to the study; 89 patients were randomly allocated cidofovir and 91 were assigned imiquimod. At the post-treatment assessment visit, a complete response had been achieved by 41 (46%; 90% CI 37·0–55·3) patients allocated cidofovir and by 42 (46%; 37·2–55·3) patients assigned imiquimod. After 6 weeks of treatment, 156 (87%) patients (78 in each group) had adhered to the treatment regimen. Five patients in the cidofovir group and seven in the imiquimod group either withdrew or were lost to follow-up before the first 6-week safety assessment. Adverse events of grade 3 or higher were reported in 31 (37%) of 84 patients allocated cidofovir and 39 (46%) of 84 patients assigned imiquimod; the most frequent grade 3 and 4 events were pain in the vulva, pruritus, fatigue, and headache. Cidofovir and imiquimod were active, safe, and feasible for treatment of vulval intraepithelial neoplasia and warrant further investigation in a phase 3 setting. Both drugs are effective alternatives to surgery for female patients with vulval intraepithelial neoplasia after exclusion of occult invasive disease. Cancer Research UK.

Fine-tuning DNA methylation by Tet proteins The modified DNA base 5-hydroxymethylcytosine (5hmC), sometimes called the sixth base, is present in the mammalian genome where it is generated by oxidation of 5-methylcytosine (5mC; the fifth base) by enzymes of the Tet family. Four papers in this issue, from the Helin, Zhang, Rao and Reik laboratories, respectively, report on the genome-wide distribution of Tet1 and/or 5hmC in mouse embryonic stem cells using the ChIP-seq technique. Links between Tet1 and transcription regulation — both activation and repression — are revealed. Anjana Rao and colleagues also describe two alternative methods with increased sensitivity for mapping single 5hmC bases. In the associated News & Views, Nathalie Véron and Antoine H. F. M. Peters discuss what these and other recent papers reveal about the role of Tet proteins in regulating DNA methylation and gene expression. Methylation at the 5′ position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development 1 . The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells 2 , 3 , 4 . Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb , Prdm14, Dppa3 , Klf2 , Tcl1 and Zfp42 ) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA is representative of the bacterial deaminase toxin family-2 (BaDTF2), and it deaminates ssDNA cytosines without a strong sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5’ or 3’ to the target cytosine are stacked linearly and make mostly sequence non-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and contributes to the stability of SsdA as a toxin. Structure-function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering. Bacterial deaminase toxins are weapons used in interbacterial antagonism and valuable tools in genome engineering. Here, the authors show how a single-stranded DNA-specific deaminase toxin, SsdA, deaminates cytosines in a sequence context-independent fashion and its utility in base editing.

Cytosine and its derivatives are an important research topic in the fields of bioorganic chemistry, molecular biology and medicine due to their key role in the structure and function of nucleic acids. The article provides a detailed overview of the natural occurrence of cytosine, its biosynthetic and degradation pathways in living organisms, as well as its physicochemical and chemical properties. Particular attention was paid to the biological activity and therapeutic applications of cytosine derivatives, including their use in cancer, antiviral and epigenetic therapy. The analytical section describes high-performance liquid chromatography techniques as a major tool for identifying and determining cytosine and its derivatives in biological samples. Examples of separation conditions, column selection, mobile phases and detection parameters for these compounds are presented. The article also provides chemical structures, graphs, comparative tables and an up-to-date review of the scientific literature, presenting a comprehensive overview of the topic, including biological, chemical and analytical aspects.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.

Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7, a recently developed cytosine deaminase, exhibits high activity across a broad protospacer range but induces unintended off-target effects, including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here, we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this, we engineer two Sdd7 variants, Sdd7e1 and Sdd7e2, enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing, narrowed editing windows, and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity, nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2, especially when delivered via eVLP, as high-fidelity CBEs poised for safe, precise therapeutic genome editing. CRISPR base editors enable precise DNA changes but often cause off-target edits. Here, authors engineer two Sdd7 variants that minimize bystander and off-target mutations and show enhanced precision when delivered as ribonucleoproteins via engineered virus-like particles.