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Novel genome editing technologies have revolutionized our ability to probe and edit the genome at a single nucleotide resolution. A major bottleneck today is the tradeoff between specificity in delivery and DNA payload size. Some of these methods are site-specific but are limited by carrying capacity and interchangeability between different targets. Other methods have a large carrying capacity, but are random in site and number of integrations. Specifically, PiggyBac transposase (PBase) is an enzyme capable of transposing mobile genetic elements up to 200 kb. However, PBase integrates into ‘TTAA’ locations which are abundant in the genome, resulting in a lack of site-specificity. On the other hand, CRISPR/Cas9 can be programmed to target specific genomic loci. Here, we engineered a novel PBase and Cas9 system that can preferentially integrate large fragments (>10 KB) to any user-specified location, combining the site-specificity of Cas9 with the integration capacity of PBase. We believe that our technology will have a transformative impact in a wide range of biomanufacturing and biomedical applications, enabling previously impossible high-value genome modifications.

ABSTRACT

Site-Specific Genome Integration

of Large DNA Fragments

Yesh Doctor and Kshitij Sachan are Juniors at Plano East Senior Highschool 

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