Selection of chromosomal DNA libraries using a multiplex CRISPR system

Abstract

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

DOI: http://dx.doi.org/10.7554/eLife.03703.001

eLife digest

Over the course of billions of years, natural evolution has produced new proteins and adapted existing ones so that they work better. Scientists have learned how to use the principles that underlie evolution to similarly engineer proteins in the laboratory. This process, known as directed evolution, is a powerful tool for improving how proteins function. Directed evolution normally involves mutating the gene that encodes the protein of interest, selecting the genes that produce the most promising proteins for another round of mutation, and repeating the process until the desired protein function is achieved.

In the first step of directed evolution, a gene is usually mutated randomly in order to create a large ‘library’ of different forms of the gene. These are joined to circular pieces of DNA known as plasmids that can replicate themselves inside cells. However, the number of plasmids than can be taken up differs from cell to cell. This complicates experiments, and the ideal directed evolution experiment would have the same number of plasmids, or target genes, being delivered into each cell.

Ryan et al. have developed a new method for performing directed evolution experiments that uses a recently developed technique called the CRISPR-Cas9 system. This can make direct changes to a DNA strand such as inserting or deleting specific sequences that code for proteins. Ryan et al. used the CRISPR-Cas9 system to create multiple DNA breaks simultaneously across the genome of yeast cells, and joined ‘barcoded’ DNA or DNA for intact genes to these breaks. This avoids the need to use plasmids to introduce foreign DNA into cells. Ryan et al. have named this method the Multiplex CRISPR (or CRISPRm) system.

Having established CRISPRm, Ryan et al. tested whether it could be used to engineer improved proteins by attempting to modify a transporter protein called CDT-1. This protein transports the sugar cellobiose into yeast cells, where it can be converted into alcohol by fermentation. This is important for making biofuel from plants. After just one round of directed evolution using CRISPRm, Ryan et al. successfully isolated a form of the CDT-1 protein that increased the rate of fermentation over 10-fold; hence this CDT-1 variant could be used to increase biofuel production.

In the future, it will be important to implement multiple selection rounds with CRISPRm, and to test how large the DNA libraries can be for directed evolution. In time, CRISPRm could find use in evolving and engineering different combinations of genes, metabolic pathways, and possibly entire genomes.

DOI: http://dx.doi.org/10.7554/eLife.03703.002