Journal article Open Access

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

Pallarès-Masmitjà, Maria; Ivančić, Dimitrije; Mir-Pedrol, Júlia; Jaraba-Wallace, Jessica; Tagliani, Tommaso; Oliva, Baldomero; Rahmeh, Amal; Sánchez-Mejías, Avencia; Güell, Marc

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  <dc:creator>Pallarès-Masmitjà, Maria</dc:creator>
  <dc:creator>Ivančić, Dimitrije</dc:creator>
  <dc:creator>Mir-Pedrol, Júlia</dc:creator>
  <dc:creator>Jaraba-Wallace, Jessica</dc:creator>
  <dc:creator>Tagliani, Tommaso</dc:creator>
  <dc:creator>Oliva, Baldomero</dc:creator>
  <dc:creator>Rahmeh, Amal</dc:creator>
  <dc:creator>Sánchez-Mejías, Avencia</dc:creator>
  <dc:creator>Güell, Marc</dc:creator>

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.</dc:description>
  <dc:source>Nature Communications 12</dc:source>
  <dc:title>Find and cut-and-transfer (FiCAT) mammalian genome engineering</dc:title>
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