Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

Landscape and Regulation of m6A and m6Am Methylome across Human and Mouse Tissues.

N6-methyladenosine (m6A), the most abundant internal mRNA modification, and N6,2'-O-dimethyladenosine (m6Am), found at the first-transcribed nucleotide, are two reversible epitranscriptomic marks. However, the profiles and distribution patterns of m6A and m6Am across human and mouse tissues are poorly characterized. Here, we report the m6A and m6Am methylome through profiling of 43 human and 16 mouse tissues and demonstrate strongest tissue specificity for the brain tissues. A small subset of tissue-specific m6A peaks can also readily classify tissue types. The overall m6A and m6Am level is partially correlated with the expression level of their writers and erasers. Additionally, the m6A-containing regions are enriched for SNPs. Furthermore, cross-species analysis revealed that species rather than tissue type is the primary determinant of methylation. Collectively, our study provides an in-depth resource for dissecting the landscape and regulation of the m6A and m6Am epitranscriptomic marks across mammalian tissues.

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Increasing the efficiency and precision of prime editing with guide RNA pairs.

The recently reported prime editor (PE) can produce all types of base substitution, insertion and deletion, greatly expanding the scope of genome editing. However, improving the editing efficiency and precision of PE represents a major challenge. Here, we report an approach termed the homologous 3' extension mediated prime editor (HOPE). HOPE uses paired prime editing guide RNAs (pegRNAs) encoding the same edits in both sense and antisense DNA strands to achieve high editing efficiency in human embryonic kidney 293T cells as well as mismatch repair-deficient human colorectal carcinoma 116 cells. In addition, we found that HOPE shows greatly improved product purity compared to the original PE3 system. We envision that this enhanced tool could broaden both fundamental research and therapeutic applications of prime editing.

Differential roles of human PUS10 in miRNA processing and tRNA pseudouridylation.

Pseudouridine synthases (PUSs) are responsible for installation of pseudouridine (Ψ) modification in RNA. However, the activity and function of the PUS enzymes remain largely unexplored. Here we focus on human PUS10 and find that it co-expresses with the microprocessor (DROSHA-DGCR8 complex). Depletion of PUS10 results in a marked reduction of the expression level of a large number of mature miRNAs and concomitant accumulation of unprocessed primary microRNAs (pri-miRNAs) in multiple human cells. Mechanistically, PUS10 directly binds to pri-miRNAs and interacts with the microprocessor to promote miRNA biogenesis. Unexpectedly, this process is independent of the catalytic activity of PUS10. Additionally, we develop a sequencing method to profile Ψ in the tRNAome and report PUS10-dependent Ψ sites in tRNA. Collectively, our findings reveal differential functions of PUS10 in nuclear miRNA processing and in cytoplasmic tRNA pseudouridylation.

Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA.

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.

Bisulfite-Free, Nanoscale Analysis of 5-Hydroxymethylcytosine at Single Base Resolution.

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

Correction to: Contrasting transcriptional responses of PYR1/PYL/RCAR ABA receptors to ABA or dehydration stress between maize seedling leaves and roots.

Following publication of the original article [1], a reader spotted that the article appears to have some misplaced/duplicated figures. In particular, Fig. 5a and Fig. 6a appear to be identical, and do not match what is written in the text. The authors apologized for this oversight and supplied the original pictures, which are reproduced below.

Contrasting transcriptional responses of PYR1/PYL/RCAR ABA receptors to ABA or dehydration stress between maize seedling leaves and roots.

BACKGROUND: The different actions of abscisic acid (ABA) in the aboveground and belowground parts of plants suggest the existence of a distinct perception mechanism between these organs. Although characterization of the soluble ABA receptors PYR1/PYL/RCAR as well as core signaling components has greatly advanced our understanding of ABA perception, signal transduction, and responses, the environment-dependent organ-specific sensitivity of plants to ABA is less well understood. RESULTS: By performing real-time quantitative PCR assays, we comprehensively compared transcriptional differences of core ABA signaling components in response to ABA or osmotic/dehydration stress between maize (Zea mays L.) roots and leaves. Our results demonstrated up-regulation of the transcript levels of ZmPYLs homologous to dimeric-type Arabidopsis ABA receptors by ABA in maize primary roots, whereas those of ZmPYLs homologous to monomeric-type Arabidopsis ABA receptors were down-regulated. However, this trend was reversed in the leaves of plants treated with ABA via the root medium. Although the mRNA levels of ZmPYL1-3 increased significantly in roots subjected to polyethylene glycol (PEG)-induced osmotic stress, ZmPYL4-11 transcripts were either maintained at a stable level or increased only slightly. In detached leaves subjected to dehydration, the transcripts of ZmPYL1-3 together with ZmPYL5, ZmPYL6, ZmPYL10 and ZmPYL11 were decreased, whereas those of ZmPYL4, ZmPYL7 and ZmPYL8 were significantly increased. Our results also showed that all of the evaluated transcripts of PP2Cs and SnRK2 were quickly up-regulated in roots by ABA or osmotic stress; conversely they were either up-regulated or maintained at a constant level in leaves, depending on the isoforms within each family. CONCLUSIONS: There is a distinct profile of PYR/PYL/RCAR ABA receptor gene expression between maize roots and leaves, suggesting that monomeric-type ABA receptors are mainly involved in the transmission of ABA signals in roots but that dimeric-type ABA receptors primarily carry out this function in leaves. Given that ZmPYL1 and ZmPYL4 exhibit similar transcript abundance under normal conditions, our findings may represent a novel mechanism for species-specific regulation of PYR/PYL/RCAR ABA receptor gene expression. A difference in the preference for core signaling components in the presence of exogenous ABA versus stress-induced endogenous ABA was observed in both leaves and roots. It appears that core ABA signaling components perform their osmotic/dehydration stress response functions in a stress intensity-, duration-, species-, organ-, and isoform-specific manner, leading to plasticity in response to adverse conditions and, thus, acclimation to life on land. These results deepen our understanding of the diverse biological effects of ABA between plant leaves and roots in response to abiotic stress at the stimulus-perception level.

Chemical and Biological Approaches to Interrogate off-Target Effects of Genome Editing Tools.

Various genome editing tools have been developed for programmable genome manipulation at specified genomic loci. However, it is crucial to comprehensively interrogate the off-target effect induced by these genome editing tools, especially when apply them onto the therapeutic applications. Here, we outlined the off-target effect that has been observed for various genome editing tools. We also reviewed detection methods to determine or evaluate the off-target editing, and we have discussed their advantages and limitations. Additionally, we have summarized current RNA editing tools for RNA therapy and medicine that may serve as alternative approaches for genome editing tools in both research and clinical applications.

Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors.

Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.

Mapping nucleolus-associated chromatin interactions using nucleolus Hi-C reveals pattern of heterochromatin interactions.

As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.

Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.

N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome.

N6,2'-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m6Am is lacking. Here, we report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5'-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5'-UTR m6A in the human transcriptome. Using m6Am-seq, we also find that m6Am and 5'-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m6Am-seq reveals the high-confidence m6Am and 5'-UTR m6A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.

Tissue-specific 5-hydroxymethylcytosine landscape of the human genome.

5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that regulates gene expression. Charting the landscape of 5hmC in human tissues is fundamental to understanding its regulatory functions. Here, we systematically profiled the whole-genome 5hmC landscape at single-base resolution for 19 types of human tissues. We found that 5hmC preferentially decorates gene bodies and outperforms gene body 5mC in reflecting gene expression. Approximately one-third of 5hmC peaks are tissue-specific differentially-hydroxymethylated regions (tsDhMRs), which are deposited in regions that potentially regulate the expression of nearby tissue-specific functional genes. In addition, tsDhMRs are enriched with tissue-specific transcription factors and may rewire tissue-specific gene expression networks. Moreover, tsDhMRs are associated with single-nucleotide polymorphisms identified by genome-wide association studies and are linked to tissue-specific phenotypes and diseases. Collectively, our results show the tissue-specific 5hmC landscape of the human genome and demonstrate that 5hmC serves as a fundamental regulatory element affecting tissue-specific gene expression programs and functions.

Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis.

Pseudouridine is the most frequent epitranscriptomic modification. However, its cellular functions remain largely unknown. Here, we show that pseudouridine synthase 7 (PUS7) is highly expressed in glioblastoma versus normal brain tissues, and high PUS7 expression levels are associated with worse survival in patients with glioblastoma. PUS7 expression and catalytic activity are required for glioblastoma stem cell (GSC) tumorigenesis. Mechanistically, we identify PUS7 targets in GSCs through small RNA pseudouridine sequencing and show that pseudouridylation of PUS7-regulated transfer RNA is critical for codon-specific translational control of key regulators of GSCs. Moreover, we identify chemical inhibitors for PUS7 and show that these compounds prevent PUS7-mediated pseudouridine modification, suppress tumorigenesis and extend the life span of tumor-bearing mice. Overall, we identify an epitranscriptomic regulatory mechanism in glioblastoma and provide preclinical evidence of a potential therapeutic strategy for glioblastoma.