-
Science (New York, N.Y.) Jul 2020CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA...
CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.
Topics: Adenine; Adenosine Deaminase; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cryoelectron Microscopy; DNA; Deamination; Escherichia coli Proteins; Gene Editing
PubMed: 32732424
DOI: 10.1126/science.abb1390 -
Nature Biotechnology Sep 2019Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted...
Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
Topics: Adenosine Deaminase; Animals; Base Sequence; CRISPR-Cas Systems; Cell Line; Directed Molecular Evolution; Gene Editing; Gene Expression Regulation, Enzymologic; Gene Targeting; Humans; INDEL Mutation; Mice
PubMed: 31332326
DOI: 10.1038/s41587-019-0193-0 -
Frontiers in Immunology 2022
Topics: Humans; Adenosine Deaminase; Intercellular Signaling Peptides and Proteins; Vasculitis; Polyarteritis Nodosa
PubMed: 36569902
DOI: 10.3389/fimmu.2022.1108853 -
Nature Jul 2022Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including...
Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans and mice; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1 mice). Adar1 mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1 mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
Topics: Adenosine Deaminase; Animals; Apoptosis; Caspase 8; Interferon Type I; Mice; Mutation; Necroptosis; RNA, Double-Stranded; RNA-Binding Proteins
PubMed: 35859176
DOI: 10.1038/s41586-022-04878-9 -
Nature Cell Biology Aug 2022Cellular senescence plays a causal role in ageing and, in mice, depletion of p16-expressing senescent cells delays ageing-associated disorders. Adenosine deaminases...
Cellular senescence plays a causal role in ageing and, in mice, depletion of p16-expressing senescent cells delays ageing-associated disorders. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16 upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16. Hence, downregulation of ADAR1 and SIRT1 mediates p16 upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16 expression.
Topics: Adenosine Deaminase; Animals; Autophagy; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Down-Regulation; Female; Humans; Mice; RNA Editing; RNA Processing, Post-Transcriptional; RNA, Messenger; Sirtuin 1
PubMed: 35851616
DOI: 10.1038/s41556-022-00959-z -
Cell Jun 2023In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid...
In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.
Topics: Animals; Proteome; Octopodiformes; RNA Editing; Temperature; Nervous System; Adenosine Deaminase; RNA
PubMed: 37295402
DOI: 10.1016/j.cell.2023.05.004 -
Circulation Research Feb 2023Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular...
BACKGROUND
Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular inflammation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation.
METHODS
Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development/dissection. Ang II (Angiotensin II) infusion of ApoE-/- mouse model combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation/dissection. The relevance of macrophage ADAR1 to human AAA was examined using human aneurysm specimens. Moreover, a novel humanized AAA model was established to test the role of human macrophages in aneurysm formation in human arteries.
RESULTS
Allograft transplantation of wild-type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that nonvascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence and severity of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB (nuclear factor kappa B) signaling, and enhanced the expression of a number of anti-inflammatory microRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 (DiGeorge syndrome critical region 8) interaction, and consequently inhibited pri- to pre-microRNA processing of microRNAs targeting IKKβ, resulting in an increased IKKβ (inhibitor of nuclear factor kappa-B) expression and enhanced NF-κB signaling. Significantly, ADAR1 was induced in macrophages and interacted with Drosha in human AAA lesions. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries.
CONCLUSIONS
Macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism, that is, Drosha protein degradation, which inhibits the processing of microRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation in AAA.
Topics: Humans; Mice; Animals; NF-kappa B; MicroRNAs; I-kappa B Kinase; Macrophage Activation; Mice, Knockout; RNA-Binding Proteins; Aortic Aneurysm, Abdominal; Aorta, Abdominal; Inflammation; Angiotensin II; Disease Models, Animal; Mice, Inbred C57BL; Adenosine Deaminase
PubMed: 36688311
DOI: 10.1161/CIRCRESAHA.122.321722 -
Immunity May 2023Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to...
Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.
Topics: RNA; Interleukin-6; Endothelial Cells; Cytokine Receptor gp130; Endothelium; Adenosine Deaminase
PubMed: 37100060
DOI: 10.1016/j.immuni.2023.03.021 -
Nature Biotechnology Feb 2022CRISPR-Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is...
CRISPR-Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is challenging. We have identified and characterized an ultrasmall family of Cas13b proteins-Cas13bt-that can mediate mammalian transcript knockdown. We have engineered compact variants of REPAIR and RESCUE RNA editors by functionalizing Cas13bt with adenosine and cytosine deaminase domains, and demonstrated packaging of the editors within a single adeno-associated virus.
Topics: Adenosine; Adenosine Deaminase; Animals; CRISPR-Cas Systems; Gene Editing; Mammals; RNA; RNA Editing
PubMed: 34462587
DOI: 10.1038/s41587-021-01030-2 -
Journal of Clinical Immunology May 2023Deficiency of adenosine deaminase 2 (DADA2), an autosomal recessive autoinflammatory disorder caused by biallelic loss-of-function variants in adenosine deaminase 2...
PURPOSE
Deficiency of adenosine deaminase 2 (DADA2), an autosomal recessive autoinflammatory disorder caused by biallelic loss-of-function variants in adenosine deaminase 2 (ADA2), has not been systemically investigated in Chinese population yet. We aim to further characterize DADA2 cases in China.
METHODS
A retrospective analysis of patients with DADA2 identified through whole exome sequencing (WES) at seventeen rheumatology centers across China was conducted. Clinical characteristics, laboratory findings, genotype, and treatment response were analyzed.
RESULTS
Thirty patients with DADA2 were enrolled between January 2015 and December 2021. Adenosine deaminase 2 enzymatic activity was low in all tested cases to confirm pathogenicity. Median age of disease presentation was 4.3 years and the median age at diagnosis was 7.8 years. All but one patient presented during childhood and two subjects died from complications of their disease. The patients most commonly presented with systemic inflammation (92.9%), vasculitis (86.7%), and hypogammaglobinemia (73.3%) while one patient presented with bone marrow failure (BMF) with variable cytopenia. Twenty-three (76.7%) patients were treated with TNF inhibitors (TNFi), while two (6.7%) underwent hematopoietic stem cell transplantation (HSCT). They all achieved clinical remission. A total of thirty-nine ADA2 causative variants were identified, six of which were novel.
CONCLUSION
To establish early diagnosis and improve clinical outcomes, genetic screening and/or testing of ADA2 enzymatic activity should be performed in patients with suspected clinical features. TNFi is considered as first line treatment for those with vascular phenotypes. HSCT may be beneficial for those with hematological disease or in those who are refractory to TNFi.
Topics: Humans; Adenosine Deaminase; Intercellular Signaling Peptides and Proteins; Cohort Studies; Retrospective Studies; Mutation
PubMed: 36807221
DOI: 10.1007/s10875-023-01432-8