ENGINEERED MULTI-SEGMENTED RNA VIRUSES FOR LARGE-SCALE COMBINATORIAL GENETIC SCREENING

­ENGINEERED MULTI-SEGMENTED RNA VIRUSES FOR LARGE-SCALE COMBINATORIAL GENETIC SCREENING

Researchers at UCSF and the Chan Zuckerberg Biohub have engineered multi-segmented RNA viruses for use in generating large-scale combinatorial libraries.

Targeted genetic screening, for example using CRISPR-based systems, is useful to dissect biological pathways. In some cases multiple genes are targeted at the same time, which can be useful in dissecting genetic pathways where a phenotype or output is only readily observable when two or more genes have been targeted. However, it can be difficult to generate libraries that comprise a comprehensive combination of targeting molecules (e.g., sgRNAs) whose identities can be readily determined following the screen.

Stage of Research

The inventors have developed engineered RNA viruses that allow for the generation of large-scale combinatorial genetic screening. The inventors exploit a tripartite recombinant lymphocytic choriomeningitis virus (LCMV) in which guide RNA (i.e., sgRNA) are attached to viral genes. By linking a sequence encoding a protein required for viral replication to the sequence encoding the guide RNA, the virus may package multiple RNA segments, allowing reassortment of guide RNAs at a high multiplicity of infection (MOI). Moreover, a recombinant virus may feature multiple guide RNAs attached to different viral genes, forcing higher levels of reassortment. These engineered viruses are then introduced into cells containing a sgRNA-guided enzyme, for example a CRISPR-associated protein (Cas), for genetic screening. Importantly, self-cleaving ribozyme sequences punctuate viral genes and sgRNA sequences, so that once the RNA segment is transcribed in the cell a functional gRNA is produced which targets complementary DNA when complexed with a Cas protein (e.g., Cas9). The output of the screening assay can be tracked using nucleotide sequencing of virions.

Applications

  • Generate large populations of diverse virions that express different nucleotide sequences, useful for generating large-scale combinatorial genetic screening libraries

Advantages

  • Many combinations of heterologous sequences can be made by combining selection of a virus-packaged set of RNA segments with the dynamics of viral infection and reassortment
  • Populations of virions can be generated using any method commonly used for viral expression in cells
  • Compatible with all CRISPR system modes of action (e.g., CRISPRi or CRISPRa), and Cas enzyme can be either expressed in cells or encoded by the virus
  • System can be applied to other viruses that support multipartite genomes, like other arenaviruses

Stage of Development

Research – in vitro

Related Web Links

https://derisilab.ucsf.edu/

Keywords

CRISPR, CRISPRi, Cas9, Integrated Screening System, Nuclease

Technology Reference

Chan Zuckerberg CZB-158F; UCSF SF2020-197

Patent Information:
For Information, Contact:
Maureen Sheehy
General Counsel
CZ Biohub
ip@czbiohub.org
Inventors:
Joseph DeRisi
Hanna Retallack
Keywords:
Cas9
CRISPR
CRISPRi
Integrated Screening System
Nuclease