Precise genome editing

Aim

Optimize methods for precise genome editing

Brief project outline

Precise genome editing based on homology directed repair (HDR) is one of the most important applications of CRISPR. However, due to the low frequency of HDR in comparison to indel (insertion or deletion) forming non homologous end joining (NHEJ), it is very challenging to perform HDR without sib selection and/or large scale screening of single cell clones. Increasing the efficiency of precise HDR is critical for expediting the success of precise genome editing projects such as SNP modification in a timely manner. Previous research by other groups have shown that using cell cycle synchronization, small molecule HDR enhancers, and RNP: ssODN covalent labelling methods increases the HDR efficiency to varying extents. Additionally, base editing using enzymes incapable of producing indels has shown to be more efficient than HDR for some targets. In this project, we will evaluate these methods separately or in combination in different targets and cells to optimize the method for precise genome editing.

Genomics-based innovative aspect of proposal

Optimizing CRISPR recombination efficiency using the Cas9 (fusion) proteins in a number of different cell types using different approaches including cell cycle synchronization and small molecule HDR enhancer. 

Broad applicability of the technique

A few research groups at UQ have shown interest in our Cas9 fusion proteins for generating CRISPR mice models. We aim to release optimised protocols for both purification of Cas9 fusion proteins with activity testing as well as an efficient CRISPR technique in cells.