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

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.

CRISPR-gRNA Design.

Gene editing has great therapeutic impact, being of interest for many scientists worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has been adapted for gene editing to serve as an efficient, rapid, and cost-effective tool. To fulfill CRISPR experiment's goals, two components are important: an endonuclease and a gRNA. The most commonly used endonucleases are Cpf1 and Cas9 and are described in depth in this chapter. The gRNA targets the genome site to be edited, giving great importance to its design to obtain increased efficiency and decreased off-target events. In this chapter, we describe different tools to design suitable gRNAs for a variety of experimental purposes.