Using computer-aided design to develop a CRISPR/Cas9-based approach to cause thousands of mutations and map their effects to the mutated genes.
The generation of large collections of mutant bacterial strains is limited due to low mutagenic efficiencies and the difficulty of tracking diverse types of mutations or their combinations. Researchers at the University of Colorado in Boulder and their collaborators have taken advantage of the high editing efficiency of the CRISPR (clustered regularly interspaced short palindromic repeats) -Cas9 system, combined with synthetic bar-codes, to develop a method that can mutate thousands of genes and easily track the mutated genes to determine their effect on the bacterial physiology.
This new editing technique, for the first time, makes it possible to induce individual mutations throughout a bacterial genome in parallel, and associate each mutation with the resulting phenotype at single-nucleotide resolution in a single experiment. This method gives researchers the ability to design and modify microorganisms in a genome-wide manner allowing them to engineer new metabolic pathways for the production of biofuels and other relevant industrial products.
A CRISPR-enabled trackable genome engineering (CREATE) cassette was developed to include a targeting guide RNA (gRNA), a DNA sequence homologous to a given target locus in the genome, and a unique bar code to tack each mutation. A computationally designed library of over 50,000 CREATE cassettes targeting multiple genome locations was synthesized and used to induce specific mutations in a bacterial population. The resulting mutant strains were tracked by genomic sequencing showing an average editing efficiency of 70%. The CREATE library was tested on a bacterial culture under thermal stress and several hundred mutants that had previously been identified as adaptations to heat were also identified with CREATE, in addition to 140 new mutations in genes involved in the bacterial response to high temperature. Furthermore, several strains that showed high stress tolerance were the result of combinations of two or more single-nucleotide mutations that would not have been detected in normal mutagenesis experiments. The potential of CREATE to identify improved mutant strains can be used to develop new and enhanced biosynthetic abilities for the biological production of fuels and relevant chemicals.
Contacts (BER PM)
Biological and Environmental Research
Department of Chemical and Biological Engineering
University of Colorado
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0008812. The authors also acknowledge support from the CAPES foundation.
Andrew Garst, Marcelo Bassalo, Gur Pines, Sean Lynch, Andrea Halweg-Edwards, Rongming Liu, Liya Liang, Zhiwen Wang, Ramsey Zeitoun, William Alexander, and Ryan Gill, “Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.” Nature Biotechnology 35, 48 (2017). [DOI: 10.1038/nbt.3718] (Reference link)
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