| Title: |
Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e |
| Authors: |
Arantes, Pablo R; Chen, Xiaoyu; Sinha, Souvik; Saha, Aakash; Patel, Amun C; Sample, Matthew; Nierzwicki, Łukasz; Lapinaite, Audrone; Palermo, Giulia |
| Source: |
Nucleic Acids Research, vol 52, iss 22 |
| Publisher Information: |
eScholarship, University of California |
| Publication Year: |
2024 |
| Collection: |
University of California: eScholarship |
| Subject Terms: |
3101 Biochemistry and Cell Biology (for-2020); 31 Biological Sciences (for-2020); Human Genome (rcdc); Biotechnology (rcdc); Genetics (rcdc); 2.1 Biological and endogenous factors (hrcs-rac); Generic health relevance (hrcs-hc); 3 Good Health and Well Being (sdg); Gene Editing (mesh); CRISPR-Cas Systems (mesh); CRISPR-Associated Protein 9 (mesh); DNA (mesh); Protein Multimerization (mesh); Humans (mesh); Adenosine Deaminase (mesh); Protein Domains (mesh); Deamination (mesh); Molecular Dynamics Simulation (mesh); Adenine (mesh); 05 Environmental Sciences (for); 06 Biological Sciences (for); 08 Information and Computing Sciences (for); Developmental Biology (science-metrix); 34 Chemical sciences (for-2020); 41 Environmental sciences (for-2020) |
| Subject Geographic: |
13931 - 13944 |
| Description: |
CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools. |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
unknown |
| Relation: |
qt8mx3c60v; https://escholarship.org/uc/item/8mx3c60v; https://escholarship.org/content/qt8mx3c60v/qt8mx3c60v.pdf |
| DOI: |
10.1093/nar/gkae1066 |
| Availability: |
https://escholarship.org/uc/item/8mx3c60v; https://escholarship.org/content/qt8mx3c60v/qt8mx3c60v.pdf; https://doi.org/10.1093/nar/gkae1066 |
| Rights: |
CC-BY-NC |
| Accession Number: |
edsbas.F01A6DC1 |
| Database: |
BASE |