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

Maria Pallarès-Masmitjà, Dimitrije Ivančić, Júlia Mir-Pedrol, Jessica Jaraba-Wallace, Tommaso Tagliani, Baldomero Oliva, Amal Rahmeh, Avencia Sánchez-Mejías () and Marc Güell ()
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Maria Pallarès-Masmitjà: Pompeu Fabra University
Dimitrije Ivančić: Pompeu Fabra University
Júlia Mir-Pedrol: Pompeu Fabra University
Jessica Jaraba-Wallace: Pompeu Fabra University
Tommaso Tagliani: Pompeu Fabra University
Baldomero Oliva: Pompeu Fabra University
Amal Rahmeh: Pompeu Fabra University
Avencia Sánchez-Mejías: Pompeu Fabra University
Marc Güell: Pompeu Fabra University

Nature Communications, 2021, vol. 12, issue 1, 1-9

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-27183-x

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