New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genomics

Background:

• CRISPR interference (CRISPRi) screening utilizes single-molecular guide RNA (sgRNA) libraries to identify genes influencing specific phenotypes.
• Traditional CRISPRi screening involves large sgRNA libraries with approximately 10^12 random sequences.

Purpose of the Study:

• To investigate the feasibility of shortening the target recognition sequence (TRS) in sgRNAs for CRISPRi screening.
• To develop a more cost-effective and efficient method for phenotype-based target gene identification.

Main Methods:

• Demonstrated that a 9-nucleotide TRS (N9) is sufficient for dCas9-mediated gene repression in Escherichia coli.
• Constructed random sgRNA plasmid libraries with shortened TRS lengths.
• Screened libraries to identify genes involved in xylose metabolism and violacein pigment production.

Main Results:

• Identified target genes for xylose metabolism using Sanger sequencing of sgRNA plasmids from Xyl- phenotypic cells.
• Discovered 17 target genes that enhance violacein production in synthetic E. coli, including seven known to increase l-tryptophan precursor pools.
• Confirmed significant violacein production increases in cells with single deletions of these identified target genes.

Conclusions:

• Shortened random TRS libraries for CRISPRi screening are effective and simpler than traditional methods.
• This approach offers a cost-effective strategy for phenotype-based target gene discovery.
• The method successfully identified genes regulating metabolic pathways and pigment production.