Recently, editing independent on DSBs 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. Cells repair these DSB through one of two major pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR) 3. Traditionally, gene editing was based on the design of artificial endonucleases that induce a double-strand break (DSB) into the sequence of interest in the genome 2.

Human gene editing technologies have significantly progressed over the last few years by the development of new editing tools 1. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. 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. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. 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). While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing.

How to get core fragments