117. AAV-TRISPR – A Novel Versatile AAV Vector Kit for Combinatorial CRISPR and RNAi Expression

The CRISPR/Cas9 system currently revolutionizes the gene therapy field due to the unique ease and efficiency with which it can be exploited for targeted DNA editing in mammalian cells. It only requires a small g(uide)RNA that directs the Cas9 protein to a specific DNA sequence which is then cleaved by Cas9. In the absence of a homologous exogenous template, the ensuing double-stranded DNA break is repaired by non-homologous end joining, an error-prone cellular mechanism frequently resulting in insertions or deletions that disrupt the original sequence. While useful in principle to perturb the expression of disease-associated genes, rapidly evolving targets such as human viruses will require concurrent expression of multiple gRNAs to thwart adverse mutational escape. To fill in this gap, we created novel highly customizable AAV vectors permitting multiplexing of up to three gRNAs in a single self-complementary vector backbone. Notably, our constructs readily accommodate and deliver minimal expression cassettes only consisting of gRNA(s) and promoter(s) with total sizes as little as 250 bp, which reduces the amount of ectopic DNA and improves vector safety. We demonstrated the power of our vectors by their use for simultaneous knock-out of three key players in apoptosis signaling, exemplifying their usefulness for dissection of cellular pathways. In addition, we engineered corresponding AAV backbones for shRNA expression, to be able to eventually juxtapose targeting on the DNA (CRISPR) and the RNA (RNAi) level in a single vector. Therefore, we made all constructs compatible with Golden Gate Assembly which minimizes work while maximizing flexibility, in turn paving the way for high-throughput cloning and screening of combinatorial RNAi/CRISPR libraries. Notably, the vectors offer three different RNA polymerase III promoters – U6, H1 or 7SK – for expression of gRNA or shRNA, thus allowing users to fine-tune g/shRNA levels and to alleviate toxic effects from over-expression. Finally, we also assembled a library of promoters for Cas9 expression comprising viral, synthetic and mammalian variants, to further expand the options for customization of our AAV/TRISPR system according to specific targets and needs. Indeed, as described in more detail in a separate abstract (Borner et al.), the selection of optimal gRNA/Cas9 vector designs even permits potent disruption of very challenging targets, such as integrated proviral HIV-1 DNA in T cells. Likewise, the use of proper regulatory elements is crucial for potent in vivo CRISPR-mediated targeting of endogenous genes in the mouse liver, as noted for the miR-122 microRNA. Considering the high degree of versatility of our vector designs, as well as the additional options for in vivo re-targeting through AAV capsid engineering, we expect our novel AAV-TRISPR kit for combinatorial gRNA/shRNA expression to substantially foster future efforts to co-exploit RNAi and CRISPR, and to benefit a multitude of human gene therapy applications.