Saturation mutagenesis genome engineering of infective FX174 bacteriophage via unamplified oligo pools and golden gate assembly

Matthew S. Faber; James T. Van Leuven; Martina M. Ederer; Yesol Sapozhnikov; Zoë L. Wilson; Holly A. Wichman; Timothy A. Whitehead; Craig R. Miller

Advances in the technologies for generating mutant libraries, synthesizing DNA, and for the assembly of large DNA fragments (van Dolleweerd et al. 2018 ) allow for the construction and assembly of user-defined mutagenesis of long nucleic acids. Nicking mutagenesis (NM) can be used to construct comprehensive single-site or other user-defined mutant libraries (Wrenbeck et al. 2016 ) using dsDNA as a template. In NM, oligos encode the desired mutations by mismatch with the parental template. On-chip ink-jet printed oligo pools have been integrated into NM (Medina-Cucurella et al. 2019) , so one can now construct heterogeneous libraries of oligos that contain tens to hundreds of thousands of high fidelity, unique sequences (Kosuri and Church 2014, Medina-Cucurella et al. 2019 ) with a low per base pair cost.