scholarly journals The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

2021 ◽  
Author(s):  
Aleksia Barka ◽  
Kiara N. Berríos ◽  
Peter Bailer ◽  
Emily K. Schutsky ◽  
Tong Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Strong Dependence ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Dna And Rna ◽  
Dna Backbone ◽  
Mechanistic Basis ◽  
Cellular Dna ◽  
Editing Enzyme

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.

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

2020 ◽  
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.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

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

2021 ◽  
Author(s):  
Aleksia Barka ◽  
Kiara Berríos ◽  
Peter Bailer ◽  
Emily Schutsky ◽  
Tong Wang ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Base Editing ◽  
Dna And Rna ◽  
Editing Enzyme

scholarly journals Comprehensive analysis of prime editing outcomes in human embryonic stem cells

2021 ◽  
Author(s):  
Omer Habib ◽  
Gizem Habib ◽  
Gue-ho Hwang ◽  
Sangsu Bae
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Cytidine Deaminase ◽  
Embryonic Stem ◽  
Cell Types ◽  
Great Promise ◽  
Nucleotide Substitutions ◽  
Base Editing

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

scholarly journals Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification

2021 ◽  
Vol 12 ◽  
Author(s):  
Marina Alexeeva ◽  
Marivi Nabong Moen ◽  
Xiang Ming Xu ◽  
Anette Rasmussen ◽  
Ingar Leiros ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Strand Break ◽  
Immunoglobulin Gene ◽  
Dna Double Strand Breaks ◽  
Cytosine Deamination ◽  
Considerable Uncertainty ◽  
Gene Diversification ◽  
Cellular Dna

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3570
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species

iScience ◽  
2018 ◽  
Vol 6 ◽  
pp. 222-231 ◽  
Author(s):  
Weizhong Chen ◽  
Ya Zhang ◽  
Yifei Zhang ◽  
Yishuang Pi ◽  
Tongnian Gu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
Cytidine Deaminase ◽  
Base Editing ◽  
Pseudomonas Species

scholarly journals Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

2020 ◽  
Author(s):  
Peng-Fei Xia ◽  
Isabella Casini ◽  
Sarah Schulz ◽  
Christian-Marco Klask ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Genetically Modified Organisms ◽  
Cytidine Deaminase ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Clostridium Ljungdahlii ◽  
Acetogenic Bacteria ◽  
A Genome ◽  
Nucleotide Resolution

AbstractAcetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

scholarly journals CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing inKlebsiella pneumoniae

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Yu Wang ◽  
Shanshan Wang ◽  
Weizhong Chen ◽  
Liqiang Song ◽  
Yifei Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Manipulation ◽  
Cytidine Deaminase ◽  
Carbapenem Resistance ◽  
Human Pathogen ◽  
Content Type ◽  
Base Editing ◽  
Highly Efficient ◽  
Carbapenem Resistant ◽  
Recent Emergence

ABSTRACTKlebsiella pneumoniaeis a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistantK. pneumoniaehas posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistantK. pneumoniae. Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation inK. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing inK. pneumoniae. By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing inK. pneumoniaeusing a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, theblaKPC-2gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistantK. pneumoniaestrain. The development of the two editing tools will significantly facilitate the genetic engineering ofK. pneumoniae.IMPORTANCEGenetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogenK. pneumoniae. We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolatedK. pneumoniaestrains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistantK. pneumoniaestrain, elucidating that theblaKPC-2gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistantK. pneumoniaestrain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverseK. pneumoniaestrains and relevantEnterobacteriaceaespecies, such as gene characterization, drug discovery, and metabolic engineering.

scholarly journals AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19

2021 ◽  
Vol 12 ◽  
Author(s):  
Anastasia Meshcheryakova ◽  
Peter Pietschmann ◽  
Philip Zimmermann ◽  
Igor B. Rogozin ◽  
Diana Mechtcheriakova
Keyword(s):  
Cytidine Deaminase ◽  
Cell Types ◽  
Gene Signature ◽  
Patient Specific ◽  
Humoral Immune ◽  
Dna And Rna ◽  
Lymphoid Structures ◽  
Specific Mapping ◽  
Key Aspects ◽  
Editing Enzyme

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

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