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Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii
Name
nihms-1660237.pdf
Description
Accepted version
Size
1.42 MB
Format
Adobe PDF
Checksum (MD5)
907f0efffc6cf24f6b98e165c17757ce
Author(s)
Dalvie, Neil C
Leal, Justin
Whittaker, Charles A
Yang, Yuchen
Brady, Joseph R
Love, Kerry R
Love, J Christopher
Date Issued
2020
Journal
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Version
Author's final manuscript
Abstract
Copyright © 2019 American Chemical Society. There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.
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DOI of Published Version
10.1021/ACSSYNBIO.9B00372
