gDesigner

gDesigner designs orthogonal synthetic guide RNAs (gRNAs) for CRISPR interference (CRISPRi) to enable programmable modulation of endogenous and synthetic promoter activity.

Key Features:

• Automated gRNA Selection: Automates selection of orthogonal gRNAs to minimize off-target effects and reduce promoter crosstalk.
• Input Flexibility: Accepts input DNA sequences in FASTA format.
• Optimization of CRISPRi Systems: Optimizes modulation of promoter activity using CRISPRi-based transcription factors.
• Integration with Cas12a Repression System: Integrates with a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates gene expression via steric hindrance of target promoters.

Scientific Applications:

• Mammalian synthetic promoter design: Expands the toolkit for designing mammalian synthetic promoters with programmable repression.
• Library generation: Enables creation of libraries of orthogonal synthetic dCas12a-repressed promoters.
• Multiplex perturbation studies: Facilitates multiplex gene perturbation using orthogonal gRNAs paired with dLbCas12a repression.
• Functional genomics and network analysis: Supports dissection of complex cellular phenotypes and exploration of gene interaction networks.

Methodology:

Computational steps include screening candidate gRNAs for orthogonality and computational minimization of off-target binding to identify gRNAs with high specificity.