AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

Abstract

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.