Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V

Abstract

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.

Keywords: CRISPR; Cas9; Streptococcus pneumoniae; genome editing; plasmids.

FIG 1

pDS05 is a temperature-sensitive plasmid that can be used for CRISPR-Cas9 genome editing in S. pneumoniae. (a) Schematic representation of plasmid pDS05. (b) Microscopy analysis of strain VL3655 [D39V(pDS05]. Overlay of GFP signals with phase-contrast image shows GFP expression. Note that the levels of fluorescence have been adjusted because of the large cell-to-cell variability in fluorescence (some cells appear dark but actually produce above-background levels of fluorescence). Preculture grown at 28°C with erythromycin (T = 0 h), (c) Images are shown of cells grown for 8 h as exponentially growing cells (balanced growth) under four different conditions: 28°C with erythromycin, 28°C without erythromycin, 40°C with erythromycin, and 40°C without erythromycin. (d) Quantification of mean fluorescence intensity of GFP of cells grown under four different conditions: 28°C with erythromycin, 28°C without erythromycin, 40°C with erythromycin, and 40°C without erythromycin at time points of 0, 2, 4, 6, and 8 h after dilution from the 28°C with erythromycin condition. Fluorescence microscopy of approximately 1,000 cells per condition per time point were quantified and analyzed using MicrobeJ and BactMAP and plotted as box plots (box size and line represent the average intensity per cell) (see Materials and Methods). The green dotted horizontal lines indicate the mean fluorescence of cells from the preculture harboring pDS05.

FIG 2

Workflow of sgRNA cloning. (a) pDS05 was designed to facilitate easy replacement of gfp by the spacer sequence of the desired sgRNA with golden gate cloning, allowing also for detection of false-positive transformants. gfp, which encodes a green fluorescent protein, is in place of the spacer sequence of sgRNA and flanked by BsaI sites. E. coli with pDS05 produces green fluorescent colonies. (b) BsaI digestion of the vector exposes 4-nucleotide (nt) overhangs. (c) For each sgRNA, forward and reverse oligonucleotides were designed, as a reverse complement of each other, which after being annealed together, contained the 20-bp spacer sequence and 4-nt overhangs that can be specifically annealed with the digested vector. (d) Ligation of the digested vector with the sgRNA annealed product was transformed into E. coli, producing white colonies.

FIG 3

Workflow for markerless deletions. (a) Uptake of the plasmid by the strain with the sgRNA sequence for the desired deletion. (b) Transformation of a homology recombination (HR) template consisting of a fusion between the upstream and downstream regions of the deletion target. (c) Plating transformants with Zn²⁺ to induce expression of Cas9. Only the cells that have taken up and integrated the HR template eliminating the recognition target are able to survive.

FIG 4

Genome editing in S. pneumoniae using CRISPR-Cas9. (a) Schematic representation of strain VL3656. The lacZ gene has been inserted downstream of the capsule operon, and a version of the plasmid with an sgRNA targeting lacZ has been transformed into the strain. (b) Transformation efficiency of lacZ and capsule operon deletion. The transformation efficiency was calculated by dividing the total number of cells as counted on plates without Cas9 inducer (1 mM ZnCl₂-MnSO₄) by the number of colonies, which were also green, in the presence of inducer. Control is the transformation assay of strain VL3656 in the absence of HR template DNA. (c) Efficiency of successful transformants screened for integration of the lacZ deletion when using no selection and when using the CRISPR-Cas9 system. Data represent the averages from three independent experiments (± standard errors [SEs]).

FIG 5

Genome analysis of the Δcps Δply ΔlytA triple mutant generated using CRISPR-Cas9 editing. (a) Colony PCR analysis of expected sizes for deletion of three virulence genes. Wild type (WT) versus VL3665. (b) Colony PCR analysis of three virulence gene deletions in the final strain VL3665. (c) Schematic representation of the D39V capsule operon region before deletion and after deletion, resulting in strain VL360. (d) Schematic representation of the ply region. (e) Schematic representation of the lytA region. (f) Whole-genome marker frequency analysis of strain VL3665. The number of mapped reads (gene dosage) is plotted as a function of the position on the circular chromosome. (g) Schematic representation of strain VL3665 with three virulence genes deleted. A zoom in of 10 kb upstream and downstream of the deleted regions is shown.