CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing

Abstract

CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.

Fig. 1

Overview of the CRISPR-FRT protocol. CRISPR-FRT makes use of the arrayed collection of Keio knockout mutants having a FRT-flanked kanamycin-resistance (Kan^R) cassette replacing each non-essential E. coli gene. CRISPR-FRT includes a gRNA-FRT that directs the Cas9 nuclease to bind and cut the two FRT sites. A convenient rescue template (e.g., a mutated gene from an evolved E. coli strain amplified by PCR, a plasmid-encoded gene variant, etc) recombines (dashed lines) over the homologous regions flanking the Kan^R cassette. Survivors are screened to separate Kan^R false positives (red X) from the kanamycin sensitive (Kan^S) true positives (green check) that replaced Kan^R cassette with the mutated gene

Fig. 2

Extended applications of CRISPR-FRT. a Members of a plasmid-encoded mutagenesis library (1–4…n) or PCR-amplified linear fragments containing multiple mutated versions of one gene can serve as rescue DNA and thereby be transferred to the chromosome. This allows for single-step transfer of a mutagenesis library to the genome or reconstruction of multiple mutations (red X) in the same gene by only using a single pair of PCR primers to generate the different rescuing templates that contain the desired mutations. b Mutations in essential genes can be delivered to Keio strains with Kan^R cassettes inserted into the closest neighboring non-essential gene if recombination (dashed lines) occurs beyond the mutation

Fig. 3

CRISPR-FRT allows rapid reconstruction and phenotypic characterization of generated mutants. a Mean colistin minimum inhibitory concentrations (MIC) for wild-type MG1655 and otherwise isogenic basR and basS mutants (n = 3; error bars represent the s.d.; two-sample unpaired t-test; ***p < 0.001 vs. wild type). b Mean ciprofloxacin minimum inhibitory concentrations (MIC) for wild-type BW25113 and otherwise isogenic nadC, vacJ and acrR mutants (n ≥ 3; error bars represent standard error of the mean; two-sample unpaired t-test **p < 0.01, ***p < 0.001 vs. wild type). c When treated with amikacin or ciprofloxacin the oppB (A₁₈₀E) mutant shows a significantly increased surviving persister fraction compared to the wild type (n = 3; error bars represent the s.d.; two-sided t-test; ***p < 0.001). d Mutations in mutL, mutH, uvrD and mfd can change the genomic mutation rate. While all mutants show a higher mutation rate, only the mutL (S₁₀₁R), mutL (H₂₇₀R), mutH (W₁₀₆R) and mfd (V₈₆₄A) mutants show a significantly higher mutation rate compared to the wild type (n = 24; error bars represent upper and lower limits of the 95% confidence intervals; ShinyFlan R package built-in two-sample comparison [30]; *p < 0.05; ***p < 0.001). e The envZ (L₁₁₆P) mutant exhibits enhanced growth characteristics in the presence of 5% (v/v) ethanol compared to the wild type (left panel). Both the maximal final density (right top panel) and the growth rate (right bottom panel) significantly improved compared to the wild type (n = 5; error bars represent s.d.; unpaired two-sided t-test; ***p < 0.001). n.s. non-significant

Fig. 4

A majority of E. coli essential genes are adjacent to a non-essential gene that is part of the Keio knockout collection. Gene distance is a count of the minimum number of genes that separate an essential gene from a non-essential gene

Fig. 5

The distribution of recA alleles encoded by the plasmid pool before CRISPR-FRT is similar to the distribution of recA mutants transferred to the chromosome after CRISPR-FRT. a Column scatter graph showing the distribution of recA mutants encoded by the plasmid pool before CRISPR-FRT and recA mutants on the chromosome after CRISPR-FRT. Mean proportion of mutants shown with heavy bars and standard deviation shown with light bars (n = 31). b Distribution of individual recA mutants before and after CRISPR-FRT. Plotting previously reported relative recombination function of recA mutants (circles, second y-axis) suggests deleterious mutants with <1% recombination activity are underrepresented in the original plasmid library pool