USING DNA LOOPING PROTEINS TO ENHANCE HOMOLOGY DIRECTED REPAIR IN VIVO FOLLOWING A CAS9 INDUCED DOUBLE STRAND BREAK

Abstract

Genome engineering methods that start with a CRISPR/Cas9 targeted genomic DNA double strand break proceed through cellular DNA repair mechanisms after the induction of the break. Imprecise nonhomologous end-joining (NHEJ) is useful for knockouts, but precise homology-directed repair (HDR) is necessary for gain of function changes. NHEJ tends to be more efficient, so directing the cell to knock in a precise sequence via HDR is an active area of research. The system we have designed uses a bivalent protein to recruit HDR donor DNA to the site of a specific DNA double strand break induced by a Cas9/sgRNA nuclease. Previously described leucine zipper dual-binding (LZD) proteins areused because they are small and stable. The system was designed to reduce the effort needed for screening, shorten the time required for the repair process, and/or decrease the amount of donor DNA needed, reducing potential off-target effects. We developed a model system in Saccharomyces cerevisiae to measure gene disruption and HDR frequencies in yeast that contain combinations of non-replicating donor DNA plasmid or linear DNA, expression plasmids of four LZD variants, and a plasmid expressing Cas9 and an sgRNA targeting either the AGC1 or ADE2 genes. The donor DNA includes a gene coding for G418 resistance in yeast. It also includes an INV-2 site recognized by the C-terminal DNA binding domain of LZDs adjacent to suboptimal regions of homology to the target gene. The N-terminal DNA binding domain of the LZDs recognizes an endogenous CREB site near the target gene. The desired recombinants are scored by their inability to grow on acetate as a sole carbon source (for AGC1) or their red color (ADE2), accompanied by resistance to G418. We believe that LZD enhancement can become a simple and valuable adjunct to any other method of improving the efficiency of HDR, in any system. We were able to show that the inclusion of LZD73 in recombination experiments increased the number of colonies presenting with the desired phenotype and genotype nearly eight-fold in the absence of a designed DNA break. We also provide evidence suggesting that the presence of LZD73 has a slight positive effect on the efficiency of Cas9 targeting. Desired recombinants were recovered after Cas9/sgRNA cleavage in an experiment where there was no apparent recombination in the absence of LZD73. Future work on this project includes optimization of the homologous sequences to improve background recombination so a more quantitative measure of the improvement observed in the presence of LZD proteins. This work can be transferred laterally to enhance other recombination-based methods in other organisms: the LZD proteins could be analogous to an adjuvant that increases overall efficiency.