ABSTRACT
Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.
Subject(s)
CRISPR-Cas Systems/genetics , Cytosine/metabolism , DNA Glycosylases , Gene Editing/methods , APOBEC-1 Deaminase/genetics , APOBEC-1 Deaminase/metabolism , Adenine/metabolism , Animals , Base Pairing/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Cytidine Deaminase , DNA Repair/genetics , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Escherichia coli/genetics , Guanine/metabolism , Rats , Uracil-DNA GlycosidaseABSTRACT
APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.
Subject(s)
APOBEC-1 Deaminase/pharmacology , Anti-HIV Agents/pharmacology , HIV-1/physiology , Recombinant Fusion Proteins/pharmacology , Amino Acid Sequence , Animals , Drug Evaluation, Preclinical , Escherichia coli/genetics , Genome, Bacterial , Genome, Viral , HEK293 Cells , HIV-1/drug effects , Humans , Mutagens/pharmacology , Mutation , Protein Multimerization , Rabbits , Sequence Homology, Amino Acid , Virion/drug effects , Virion/physiology , Virus AssemblyABSTRACT
While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.
Subject(s)
Aorta/drug effects , Atherosclerosis/drug therapy , Biflavonoids/therapeutic use , APOBEC-1 Deaminase , Animals , Aorta/pathology , Atherosclerosis/pathology , Biflavonoids/pharmacology , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Lipids/blood , Mice , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Phospholipases A/antagonists & inhibitors , Receptors, LDL/genetics , Receptors, LDL/metabolism , Tunica Intima/drug effects , Tunica Intima/pathologyABSTRACT
Increased serum concentrations of low density lipoproteins represent a major cardiovascular risk factor. Low-density lipoproteins are derived from very low density lipoproteins secreted by the liver. Apolipoprotein (apo)B that constitutes the essential structural protein of these lipoproteins exists in two forms, the full length form apoB-100 and the carboxy-terminal truncated apoB-48. The generation of apoB-48 is due to editing of the apoB mRNA which generates a premature stop translation codon. The editing of apoB mRNA is an important regulatory event because apoB-48-containing lipoproteins cannot be converted into the atherogenic low density lipoproteins. The apoB gene is constitutively expressed in liver and intestine, and the rate of apoB secretion is regulated post-transcriptionally. The translocation of apoB into the endoplasmic reticulum is complicated by the hydrophobicity of the nascent polypeptide. The assembly and secretion of apoB-containing lipoproteins within the endoplasmic reticulum is strictly dependent on the microsomal tricylceride transfer protein which shuttles triglycerides onto the nascent lipoprotein particle. The overall synthesis of apoB lipoproteins is regulated by proteosomal and nonproteosomal degradation and is dependent on triglyceride availability. Noninsulin dependent diabetes mellitus, obesity and the metabolic syndrome are characterized by an increased hepatic synthesis of apoB-containing lipoproteins. Interventions aimed to reduce the hepatic secretion of apoB-containing lipoproteins are therefore of great clinical importance. Lead targets in these pathways are discussed.
Subject(s)
Apolipoproteins B/genetics , Carrier Proteins/antagonists & inhibitors , Lipoproteins, VLDL/metabolism , APOBEC-1 Deaminase , Animals , Apolipoproteins B/antagonists & inhibitors , Apolipoproteins B/metabolism , Carrier Proteins/physiology , Cytidine Deaminase/genetics , Cytidine Deaminase/physiology , Fatty Acids, Omega-3/pharmacology , Humans , Hypobetalipoproteinemias/genetics , Insulin/pharmacology , Lipoproteins, LDL/blood , PPAR alpha/agonists , Proteasome Endopeptidase Complex/physiology , Protein Biosynthesis , RNA Editing , Transcription, GeneticABSTRACT
BACKGROUND & AIMS: This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine. METHODS: Apobec-1-deleted mice (APOBEC-1(-/-)) and wild-type controls were treated with 12 Gy of whole-body gamma-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3' untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3' untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays. RESULTS: After gamma-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC-1(-/-) mice. In wild-type mice treated with lipopolysaccharide before gamma-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E 2 mediated by cyclooxygenase-2. No such effect was observed in the APOBEC-1(-/-) mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3' untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways. CONCLUSIONS: Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability.
Subject(s)
Cytidine Deaminase/physiology , Gene Expression Regulation, Enzymologic , Intestines/radiation effects , Isoenzymes/genetics , Prostaglandin-Endoperoxide Synthases/genetics , Radiation Injuries/prevention & control , 3' Untranslated Regions , APOBEC-1 Deaminase , Animals , Cyclooxygenase 2 , Gamma Rays , Imidazoles/pharmacology , Intestinal Mucosa/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/physiology , Pyridines/pharmacology , RNA, Messenger/analysis , Stem Cells/radiation effects , p38 Mitogen-Activated Protein KinasesABSTRACT
We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.
Subject(s)
B-Lymphocytes/enzymology , Cytidine Deaminase/biosynthesis , Germinal Center/cytology , RNA Editing , APOBEC-1 Deaminase , Amino Acid Sequence , Animals , Apolipoproteins B , Cycloheximide/pharmacology , Cytidine Deaminase/chemistry , Cytidine Deaminase/genetics , DNA, Complementary/isolation & purification , Enzyme Induction/drug effects , Gene Library , Germinal Center/enzymology , Mice , Molecular Sequence Data , Open Reading Frames , Phylogeny , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Sequence Alignment , Tumor Cells, CulturedABSTRACT
RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.