Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Annu Rev Biochem ; 92: 43-79, 2023 06 20.
Article in English | MEDLINE | ID: mdl-37018843

ABSTRACT

DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.


Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Associated Protein 9/genetics , Genome , DNA/genetics , DNA/metabolism
2.
Chembiochem ; 24(16): e202200788, 2023 08 15.
Article in English | MEDLINE | ID: mdl-36947856

ABSTRACT

Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.


Subject(s)
Adenosine Deaminase , Gene Editing , Adenosine Deaminase/metabolism , RNA, Transfer/chemistry , DNA/genetics , Mutation , CRISPR-Cas Systems
3.
CRISPR J ; 5(2): 294-310, 2022 04.
Article in English | MEDLINE | ID: mdl-35353638

ABSTRACT

Adenine base editors (ABEs) have been subjected to multiple rounds of mutagenesis with the goal of optimizing their function as efficient and precise genome editing agents. Despite an ever-expanding data set of ABE mutants and their corresponding DNA or RNA-editing activity, the molecular mechanisms defining these changes remain to be elucidated. In this study, we provide a systematic interpretation of the nature of these mutations using an entropy-based classification model that relies on evolutionary data from extant protein sequences. Using this model in conjunction with experimental analyses, we identify two previously reported mutations that form an epistatic pair in the RNA-editing functional landscape of ABEs. Molecular dynamics simulations reveal the atomistic details of how these two mutations affect substrate-binding and catalytic activity, via both individual and cooperative effects, hence providing insights into the mechanisms through which these two mutations are epistatically coupled.


Subject(s)
Gene Editing , RNA Editing , Adenine/metabolism , CRISPR-Cas Systems , Computational Biology , RNA/genetics , RNA Editing/genetics
4.
Sci Adv ; 6(10): eaaz2309, 2020 03.
Article in English | MEDLINE | ID: mdl-32181363

ABSTRACT

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.


Subject(s)
APOBEC Deaminases/chemistry , Adenine/metabolism , Adenosine Deaminase/chemistry , DNA, Single-Stranded/chemistry , DNA/chemistry , Escherichia coli Proteins/chemistry , Gene Editing/methods , APOBEC Deaminases/genetics , APOBEC Deaminases/metabolism , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Binding Sites , Cloning, Molecular , DNA/genetics , DNA/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Humans , Kinetics , Molecular Dynamics Simulation , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thermodynamics
SELECTION OF CITATIONS
SEARCH DETAIL
...