Targeted insertional mutagenesis libraries for deep domain insertion profiling.

ABSTRACT: Domain recombination is a key principle in protein evolution and protein engineering, but inserting a donor domain into every position of a target protein is not easily experimentally accessible. Most contemporary domain insertion profiling approaches rely on DNA transposons, which are constrained by sequence bias. Here, we establish Saturated Programmable Insertion Engineering (SPINE), an unbiased, comprehensive, and targeted domain insertion library generation technique using oligo library synthesis and multi-step Golden Gate cloning. Through benchmarking to MuA transposon-mediated library generation on four ion channel genes, we demonstrate that SPINE-generated libraries are enriched for in-frame insertions, have drastically reduced sequence bias as well as near-complete and highly-redu

SUBMITTER: Coyote-Maestas W 

PROVIDER: S-EPMC6954442 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature