Journal
CELL
Volume 171, Issue 6, Pages 1453-+Publisher
CELL PRESS
DOI: 10.1016/j.cell.2017.10.034
Keywords
-
Categories
Funding
- NSF-GRFP [1122492]
- NIH Predoctoral Training Grant in Genetics [T32GM007499]
- Howard Hughes Medical Institute
- DARPA [HR0011-15-C-0091, N66001-12-C-4020]
- DOE [DE-FG02-02ER63445]
- NIH [1R01GM117230-01, 1U54CA209992-01]
- Arnold and Mabel Beckman Foundation
- Division Of Graduate Education
- Direct For Education and Human Resources [1122492] Funding Source: National Science Foundation
Ask authors/readers for more resources
We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >10(5). This method was used to diversify a heterologous beta-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available