[New methods for cloning large gene clusters based on CRISPR/cas9].

Area of Science:

• Synthetic biology
• Molecular biology
• Genomics

Background:

• Traditional cloning methods face limitations in efficiently obtaining large DNA fragments (>10 kb).
• Methods like PCR amplification, random library cloning, and restriction endonuclease digestion have significant drawbacks, including labor intensity, high mutation rates, and difficulty in site identification.
• These limitations hinder advancements in synthetic biology that require large genomic sequences.

Purpose of the Study:

• To review and classify emerging high-performance methods for cloning large genomic fragments.
• To compare the efficiency and limitations of various large fragment cloning techniques.
• To provide criteria for selecting appropriate cloning methods based on fragment size and experimental needs.

Main Methods:

• Classification of current large fragment cloning technologies.
• Discussion of genome-wide editing tools, specifically the CRISPR/Cas9 system, for targeted DNA cutting.
• Integration of CRISPR/Cas9 with DNA assembly methods such as Gibson assembly and transformation-associated recombination (TAR).

Main Results:

• CRISPR/Cas9 enables precise identification and cutting of specific DNA sequences for generating desired large fragments.
• Combining CRISPR/Cas9 with Gibson or TAR assembly significantly enhances the efficiency of cloning large DNA fragments.
• Novel genome-wide editing approaches offer a high-performance alternative to traditional cloning methods.

Conclusions:

• Advanced cloning strategies utilizing genome-wide editing and assembly technologies are crucial for synthetic biology.
• The choice of cloning method should be guided by fragment size and specific research requirements.
• CRISPR/Cas9-based methods represent a significant leap forward in efficiently cloning large genomic sequences.