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Find and cut-and-transfer (FiCAT) mammalian genome engineering

Pallares-Masmitja, Maria; Ivancic, Dimitrije; Mir-Pedrol, Julia; Jaraba-Wallace, Jessica; Tagliani, Tommaso; Oliva, Baldomero; Rahmeh, Amal; Sanchez-Mejias, Avencia; Guell, Marc

NATURE COMMUNICATIONS
2021
VL / 12 - BP / - EP /
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. Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.

AccesS level

Gold

MENTIONS DATA