Abstract
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.
Highlights
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing
Editing independent on double-strand break (DSB) has been developed with methodologies based on directly editing DNA bases with deaminases, namely base editors (BEs)[4] and in situ replacing DNA bases with aid of a reverse transcriptase, namely prime editors (PEs)[5]
Precise gene delivery methodologies based on nonhomologous end joining (NHEJ) have been developed such as homology independent targeted integration (HITI)[7]
Summary
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Reporter cell line assays showed the highest levels of programmable insertion from PB variants combining mutants with increased excision activity and decreased target DNA binding activity, which is consistent with our principle of design.
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