Columbia Technology Ventures

Method for generating diverse guide RNA libraries for CRISPR screens

This technology utilizes reprogrammed bacterial cells to efficiently and cost-effectively generate highly comprehensive, genome-wide crRNA libraries for CRISPR screens. crRNA is the canonical form of guide RNA (gRNA) that has been engineered and widely used.

Unmet Need: Efficient, economical method for producing gRNA libraries

The current method of generating gRNA libraries for genome-wide CRISPR screens is pooled oligonucleotide synthesis. This method can be costly, labor-intensive and time-consuming, with a turnaround of 1-2 weeks or longer. Furthermore, this method generates libraries with low diversity, substantially limiting the coverage and sensitivity of CRISPR screens.

The Technology: Reprogramming CRISPR-Cas machinery to generate diverse crRNA libraries

This technology reprograms the S. pyogenes CRISPR-Cas system to convert bacterial cells into “factories” that generate hundreds of thousands of crRNAs in as short as a single day. The resulting crRNA library covers up to 95% of the targeted microbial genome, with the average gene targeted by more than 100 distinct crRNAs. This method can generate both single and dual crRNA libraries to elucidate genetic interactions and record the evolutionary trajectory of acquired traits.

This technology has been validated with S. aureus, E. coli, and wild-type bacterial pathogens.

Applications:

  • Generation of highly comprehensive single and dual crRNA libraries for use in CRISPR screens
  • Research tool for recording evolutionary trajectory of acquired phenotypes (e.g. antibiotic resistance)
  • crRNA library generation in bacterial systems that are refractory to genetic manipulation (e.g. clinical isolates)

Advantages:

  • Faster turnaround (1-2 days vs. 1-2 weeks)
  • 10-100 times cheaper than alternative methods (e.g., pooled oligonucleotide synthesis)
  • Can generate single and dual crRNA libraries
  • Substantially increases crRNA library diversity
  • Achieves varying degrees of transcriptional repression critical for uncovering complex phenotypes
  • Can be applied in S. aureus, E. coli, and wild-type bacterial systems

Lead Inventor:

Saeed Tavazoie, Ph.D.

Patent Information:

Patent Pending

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