Microbial Genome CRISPR Plasmid Library Construction

Microbial genomes usually contain thousands of genes. It is particularly important to discover the correlation between these genes and phenotypes with high throughput and low cost. Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/Cas9 system. Inhibiting or activating gene expression by the genome-wide CRISPR library can help researchers analyze gene groups associated with a certain phenotype at the whole-genome level under a variety of screening conditions, providing more valuable discoveries for microbial cell factory research.

Creative Biogene offers fully customized gRNA libraries, with complete coverage and uniform distribution, for CRISPR knockout, CRISPRa, and CRISPRi. The library coverage can reach >99% with NGS validation. We have licensed Dual or All-In-One CRISPR vectors in stock. There is no sequence restriction for any projects.

High-throughput CRISPR Screens

Pooled CRISPR libraries, are ideal for high-throughput screening of important molecular targets. Screening using the CRISPR technology is particularly advantageous because of its simplicity, specificity and versatility. The genome-wide libraries were designed to target every gene in the microbial genomes, and knock-out or transcriptionally activate/inhibit each gene, respectively. Libraries are amplified, packaged and transduced into host cells to generate mutant cell lines. The mutants can then be screened under specific conditions, such as by positive or negative selection, to identify important genes in a pathway.

CRISPR/Cas9 loss-of-function screens have been used in a variety of microorganisms. All broadly used Cas9 genome-wide libraries have at least 4 single-guide RNA (sgRNA) per gene and contain over 65,000 sgRNAs. Genome-wide CRISPR/Cas9 sgRNA libraries have been iteratively optimized to reduce off-target activity and increase on-target efficiency.