Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs

Area of Science:

• Molecular Biology
• Genetics
• Synthetic Biology

Background:

• Multiplex genome editing using CRISPR-Cas9 is valuable for efficiency but faces accuracy challenges.
• Simultaneous editing of multiple genes requires precise control over guide RNA function.

Purpose of the Study:

• To develop a highly accurate method for multiplex genome editing in Escherichia coli.
• To demonstrate the efficacy of truncated single-molecular guide RNAs (sgRNAs) for simultaneous gene editing.

Main Methods:

• Utilized a 5'-end-truncated single-molecular guide RNA (sgRNA) strategy for CRISPR-Cas9 mediated genome editing.
• Applied the method to simultaneously edit two and three genes (galK, xylB, srlD) in Escherichia coli at single-nucleotide resolution.
• Tested the method on targeted editing of cI and ilvG genes in E. coli.

Main Results:

• Achieved highly efficient, single-nucleotide level simultaneous editing of galK and xylB genes.
• Successfully demonstrated simultaneous editing of three genes (galK, xylB, and srlD) with single-nucleotide resolution.
• Truncated sgRNAs enabled 30% efficiency in simultaneous editing of cI and ilvG genes, unlike untruncated sgRNAs.

Conclusions:

• The truncated sgRNA method significantly enhances accuracy and efficiency in multiplex genome editing.
• This technique allows for precise genetic modifications in Escherichia coli, with practical implications for synthetic biology.
• The developed method shows potential for broad application in creating engineered organisms.