Structural and functional insights into internal domain replacement in SpCas9 for protein engineering
Seonhong Kim1, Hyuk Won2, Jungnam Bae1
1Department of Biological Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
Scientific Reports
|November 25, 2025
Summary
Researchers engineered CRISPR-Cas9 by replacing a dispensable C-terminal region with a deaminase domain. This creates a versatile base editor with tunable editing windows for precise genome editing applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR-Cas9 is a key genome editing technology.
- Enhancing CRISPR-Cas9 requires integrating new functional domains.
- Identifying suitable insertion sites is crucial for maintaining Cas9 function.
Purpose of the Study:
- To identify a suitable region in Streptococcus pyogenes Cas9 (SpCas9) for foreign domain integration.
- To engineer a novel SpCas9 variant with base editing capabilities.
- To evaluate the efficiency and versatility of the engineered base editor.
Main Methods:
- Structural and biochemical analyses of SpCas9 variants.
- Site-directed mutagenesis to remove SpCas9 residues 1242-1263.
- Integration of the E. coli tRNA adenosine deaminase (TadA) domain.
- Functional assessment of the engineered SpCas9-TadA base editor.
Main Results:
- A C-terminal region (residues 1242-1263) of SpCas9 was identified as dispensable for its activity.
- An engineered SpCas9-TadA variant exhibited deamination efficiency comparable to ABE8e.
- The linker design offers potential for modulating the base editing window.
Conclusions:
- Targeted engineering of the identified SpCas9 region enables the development of novel genome editing tools.
- The SpCas9-TadA variant represents a promising platform for precise base editing.
- This approach facilitates the creation of more versatile and efficient CRISPR-based technologies.
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