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Updated: Oct 21, 2025
08:22
CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
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Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration.
Siqi Yang1,2, Yiwen Zhang1,2, Jiaqi Xu3
1Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Nucleic Acids Research
|September 3, 2021
Summary
Researchers engineered a new CRISPR-associated transposase (CAST) for efficient, parallel gene editing. This tool enables complex cell programming and synthetic biology applications by manipulating multiple genes simultaneously.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Genetics
Background:
- Cell engineering traditionally involves single-gene manipulation, limiting complexity.
- CRISPR-associated transposases (CASTs) offer potential for multiplexed gene manipulation.
- Orthogonal CASTs allow for parallel manipulation of different genetic loci.
Purpose of the Study:
- To characterize a novel CAST from Pseudoalteromonas translucida KMM520 (PtrCAST).
- To evaluate PtrCAST's efficiency, cargo capacity, and mismatch tolerance.
- To demonstrate orthogonal activity between PtrCAST and VchCAST for multiplexed chromosomal integration.
Main Methods:
- Characterization of PtrCAST activity and PAM preference.
- Testing insertion efficiency with larger DNA cargo.
- Demonstrating orthogonal activity with VchCAST in Escherichia coli using distinct crRNAs and mini-Tn substrates.
- Assessing multiplexed integration at specific chromosomal loci.
Main Results:
- PtrCAST exhibits high insertion efficiency for larger DNA cargo and multiplexed transposition.
- PtrCAST demonstrates tolerance to mismatches beyond the 4-nucleotide seed sequence.
- PtrCAST operates orthogonally with VchCAST, enabling parallel manipulation of distinct genetic loci.
- The MUCICAT system successfully achieved multiplexed orthogonal integration in E. coli.
Conclusions:
- PtrCAST is a versatile tool for cell engineering, offering high efficiency and flexibility.
- Orthogonal CASTs, like PtrCAST and VchCAST, enable precise, parallel manipulation of multiple genes.
- Multiplexed orthogonal MUCICAT is a powerful platform for cell programming and synthetic biology.
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