(SD2022-279) Mutant ZRANB2 zinc finger proteins with GGG RNA sequence targeting specificity

Background

Existing RNA-targeting tools for sequence-specific manipulation include anti-sense oligos (ASOs), designer PUF proteins and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems. However, there are significant limitations to each of the current tools. ASOs are usually not available for most RNA manipulations other than gene silencing. Designer proteins, such as PUF (Pumilio and FBF homology protein), possess low RNA recognition efficiency and it remains challenging to target RNA sequences >8-nucleotides (nt) in length. The bulky Cas protein (Cas13d: average 930 amino acids) leads to complication for transgene delivery and concerns of its immunogenicity due to its bacterial origin.

Mutants of zinc finger(ZnF) proteins in ZRANB2 recognize a single-strand RNA containing a novel GGG motif with micromolar affinity, compared to the original motif GGU. These mutants serve as a foundation for RNA-binding ZnF designer protein engineering for in vivo RNA sequence-specific targeting.

ZnFs are generally compact domains (~3kDa each) that have been successfully engineered for DNA recognition as modular arrays. A ZnF-based system has unique advantages, especially in a therapeutic context: (1) Broad application with the possibility to fuse with other effector domains; (2) High efficiency of RNA recognition (3 RNA bases recognized per 30-amino-acid ZnF) with a small size of protein. Only 4 ZnFs (~100 aa) is required for specific targeting in the transcriptome. (3) Humanized components without immunogenic concern.

By engineering new sequence specificity of the ZRANB2 ZnF1, researchers from UC San Diego identified 13 mutants that altered their preferred RNA binding motif from GGU to GGG. They are N24R, N24H, N14D/N24R, N14D/N24H, N14R/N24R, N14R/N24H, N14H/N24R, N14H/N24H, N14Q/N24R, N14Q/N24H, N14E/N24R, N14S/N24R, N14E/N24H.

Technology Description

Applications

The mutant ZnF proteins could be used as a component of a RNA-targeting designer array.

The ZnF arrays could be fused with RNA effector proteins for various RNA manipulations (RNA degradation, RNA splicing control, RNA translation etc.) with potential applications in both biological research and RNA-targeting therapeutics.

Only 6 ZnFs are required to target RNA specifically (18-nt) in the human transcriptome.

The designer ZnF-array size is only ~100 amino acids, which is much more convenient to use and especially beneficial in therapeutic applications considering the limited packaging capacity of vectors like adenovirus-associated virus (AAV).

Advantages

State Of Development

Intellectual Property Info

UC San Diego is securing patent rights (https://patents.google.com/patent/WO2023164549A2 ) on the technology and welcomes interest from companies in a position to commercialize this invention.

Related Materials

• Qishan Liang, Joy S. Xiang, Kevin D. Corbett, Gene Yeo, Engineering sequence specificity in an RNA-binding zinc finger protein, Biophysical Journal, Volume 121, Issue 3, Supplement 1, 2022, Pages 479a-480a - 02/11/2022