Molecular basis for discrimination of DNA and RNA deamination by the base-editing enzyme APOBEC3A

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

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Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽

10.1038/s41375-021-01282-6 ◽

2021 ◽

Author(s):

Christos Georgiadis ◽

Jane Rasaiyaah ◽

Soragia Athina Gkazi ◽

Roland Preece ◽

Aniekan Etuk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Specific Binding ◽

Base Editing ◽

Car T Cells ◽

Dna And Rna ◽

Car T ◽

T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

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Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG

Circulation Research ◽

10.1161/circresaha.120.318674 ◽

2021 ◽

Author(s):

Shuhong Ma ◽

Wenjian Jiang ◽

Xujie Liu ◽

Wen-Jing Lu ◽

Tao Qi ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Mouse Model ◽

Pathogenic Mutation ◽

Adeno Associated Virus ◽

Vitro Fertilization ◽

Base Editing ◽

Dna And Rna ◽

Hereditary Disorders

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.

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2. Vital signs: the molecules of life

Molecules: A Very Short Introduction ◽

10.1093/actrade/9780192854308.003.0002 ◽

2003 ◽

pp. 33-49

Author(s):

Philip Ball

Keyword(s):

Amino Acids ◽

Molecular Basis ◽

Vital Signs ◽

Hierarchical System ◽

Biological Cell ◽

Molecular Processes ◽

Huge Number ◽

Dna And Rna ◽

Long Chains

‘Vital signs: the molecules of life’ outlines the molecular basis of life. Understanding molecular life is as much about understanding interactions between molecules and their position within the hierarchical system as it is about identifying the actors. Proteins are a specific type of molecule that perform a huge number of processes inside a biological cell. They are formed by subunits, which in turn are formed from long chains of amino acids. The instructions for these chains are contained, transmitted, and effected using DNA and RNA, another species of long, repeating chains of molecules. The molecular processes and interactions involving these molecules are well understood, but molecular understanding will never give the full story.

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Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors

Science Advances ◽

10.1126/sciadv.aaz2309 ◽

2020 ◽

Vol 6 (10) ◽

pp. eaaz2309 ◽

Cited By ~ 4

Author(s):

Kartik L. Rallapalli ◽

Alexis C. Komor ◽

Francesco Paesani

Keyword(s):

Conformational Changes ◽

Single Mutation ◽

Base Editing ◽

Functional Roles ◽

Dna Base ◽

Dna Editing ◽

Dynamics Simulations ◽

Editing Enzyme ◽

Adenine Base

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

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Next-generation cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

10.1101/2020.02.11.944165 ◽

2020 ◽

Cited By ~ 2

Author(s):

Yi Yu ◽

Thomas Leete ◽

David A. Born ◽

Lauren Young ◽

Luis A. Barrera ◽

...

Keyword(s):

Cytidine Deaminase ◽

Point Mutations ◽

Next Generation ◽

Dna Double Strand Breaks ◽

Cytosine Deamination ◽

Base Editing ◽

Dna And Rna ◽

Fold Reduction ◽

Target Dna ◽

Target Activity

Abstract/introductory paragraphCytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T•A to C•G point mutations in the human genome without induction of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) ‘BE3’, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

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Synthetic DNA and RNA Programming

Genes ◽

10.3390/genes10070523 ◽

2019 ◽

Vol 10 (7) ◽

pp. 523

Author(s):

O’Donoghue ◽

Heinemann

Keyword(s):

Synthetic Biology ◽

Molecular Biology ◽

Molecular Basis ◽

Molecular Mechanisms ◽

Research Articles ◽

Original Research ◽

Special Issue ◽

Synthetic Dna ◽

Dna And Rna ◽

Rna And Dna

Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.

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A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants

10.1101/2021.11.10.468138 ◽

2021 ◽

Author(s):

Shaofang Li ◽

Lang Liu ◽

Wenxian Sun ◽

Xueping Zhou ◽

Huanbin Zhou

Keyword(s):

High Activity ◽

Large Scale ◽

Comprehensive Evaluation ◽

Rice Genome ◽

Plant Genome ◽

Sequencing Analysis ◽

Single Nucleotide Variants ◽

Base Editing ◽

Dna And Rna ◽

Adenine Base

The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects. In this study, we performed a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, revealed that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring the CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicated that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the plant genome. Furthermore, we detected off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tended to cluster, while off-target A>G DNA mutations rarely clustered.Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.

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Mendelian Disease caused by variants affecting recognition of Z-DNA and Z-RNA by the Zα domain of the double-stranded RNA Editing Enzyme ADAR

10.31219/osf.io/4mja3 ◽

2019 ◽

Author(s):

Alan Herbert

Keyword(s):

Double Helix ◽

Protein Isoforms ◽

Mendelian Disease ◽

Loss Of Function ◽

Double Stranded Rna ◽

Parental Chromosome ◽

Dna And Rna ◽

Left Handed ◽

Z Dna ◽

Editing Enzyme

Variants in the human double-stranded RNA (dsRNA) editing enzyme ADAR produce three well-characterized rare Mendelian Diseases: Dyschromatosis Symmetrica Hereditaria (DSH)(OMIM: 127400), Aicardi-Goutières syndrome (AGS)(OMIM: 615010) and Bilateral Striatal Necrosis/Dystonia (BSD). ADAR encodes p150 and p110 protein isoforms. p150 incorporates the Zα domain that binds left-handed Z-DNA and Z-RNA with high affinity through contact of highly conserved residues with the DNA and RNA double-helix. In certain individuals, frameshift variants on one parental chromosome in the second exon of ADAR produce haploinsufficiency of p150 while maintaining normal expression of p110. In other individuals, loss of p150 expression from one chromosome allows mapping of Zα p150 variants from the other parental chromosome directly to phenotype. The analysis reveals that loss of function Zα variants cause dysregulation of innate interferon responses to dsRNA. This approach confirms a biological role for the left-handed conformation in human disease, further validating the power of Mendelian genetics to provide unambiguous answers. The findings reveal that the human genome encodes genetic information using both shape and sequence.

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