Our scientists have engineered a novel hybrid CRISPR-based forward genetic screening platform that is based on the dual simultaneous expression of the two orthogonal Cas nucleases Cas9 & Cas12a, and the delivery of hybrid guide RNA (hgRNA) libraries. The hybrid system combines the DNA editing activity of Cas9 and Cas12a together with the RNA processing activity of Cas12a, which allows guide multiplexing by processing combined hgRNAs transcribed from a single U6 promoter into mature Cas9 and Cas12a single guide RNAs (sgRNAs). Importantly, the design of the hybrid system allows for the defined combination of target sites and selected multiplexed editing outcomes, thus allowing the systematic interrogation of exons coupled with high throughput phenotyping for the first time.
Recently, the CRISPR Cas9 system has been applied for combinatorial screening to target gene pairs or non-coding RNAs. However, these multi-targeting systems exhibit reduced efficiency due to recombination between Cas9 tracrRNAs and U6 promoters. In order to overcome these limitations, promoters from difference species and genes have been applied and mutations introduced into the tracrRNA scaffold. Although these modifications reduce recombination, their editing efficiency was still compromised. Another approach that has been used to improve combinatorial targeting has been the application of orthologous Cas9 nucleases. However, due to the utilization of multiple promoters that do not allow for targeted selection of guide pairs and random combination of guides, the applied screening libraries only comprise a very limited number of targeted genes. Furthermore, these systems ignore splice isoforms, largely due to the lack of available experimental methodologies for the combination of selected target sites.
- Significantly more sites for Cas12a can be targeted on the mammalian genome
- Highly specific combinatorial targeting of multiple genes, genetic elements or genomic sites
- Can be applied to pooled or arrayed functional genomic screens to elucidate:
- Biological functions of alternative and constitutive exons
- Study of genetic interactions and vulnerabilities
- Target paralogous genes
- More efficient than other Cas9 systems
- IP - Provisional patent application filed