Journal: Nature Communications

Article Title: Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

doi: 10.1038/s41467-025-59068-8

Figure Lengend Snippet: A Cloning strategy to produce a library of pegRNAs targeting luc from a ssDNA oligo pool. The oligo pool was amplified by PCR as well as the backbone of pVL4134 that includes the luc spacer sequence and assembled by Golden Gate digestion/ligation followed by transformation to S. pneumoniae containing luc and mbPE2 S.pn . Chromosomal DNA was isolated from colonies grown in absence or presence of aTc (to induce mbPE2 S.pn ) and PCR amplified using staggered primers followed by Illumina sequencing (see “Methods”). 2FAST2Q was used to extract and count pegRNAs and luc alleles. B Schematic overview of the pegRNA design encoding for different base pair substitutions. ( C, D ) Single base pair substitutions and multiple base pair substitutions are installed with high selection efficiency. For mutation frequencies, results of four biological replicates are plotted in box-plots, with first quartile, median, and third quartile indicated. Whiskers indicate the maximum value, at most 1.5x IQR from the first or third quartile. Outliers are plotted in red. For pegRNA frequencies, the mean of four experiments is plotted. C pegRNA abundance of - aTc samples (control, purple) and + aTc samples (orange) are shown. For reference, values of a successful edit (113 bp deletion) and unsuccessful edit (63 bp insertion) are also plotted. D The fraction of luc reads carrying the desired mutation over the total reads in that sample is shown for induced (aTc treated, orange) and non-induced (-aTc; control) samples on the left Y-axis. The average fraction of pegRNA reads for the substitutions is shown as dots on the right Y-axis. Red dots indicate outliers in induced samples. Non-induced samples only have data indicated as lines in double mutant “TCCTGAA” and triple mutant “TGCAGAA”, as only one non-zero datapoint was collected in those samples.

Article Snippet: Chromosomal DNA from the pegRNA pool screening samples was extracted for Illumina library preparation using the Promega kit as described previously .

Techniques: Cloning, Amplification, Sequencing, Ligation, Transformation Assay, Isolation, Illumina Sequencing, Selection, Mutagenesis, Control

Journal: Nature Communications

Article Title: Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

doi: 10.1038/s41467-025-59068-8

Figure Lengend Snippet: A Schematic overview of the RTT design strategy used to edit bases distal from the PAM site. Every pegRNA introduces a single PAM mutation (G to C) and a second, purine to pyrimidine (or vice versa) transversion introduced at varying distances from the PAM sequence. B Fraction of correctly edited luc alleles and pegRNA read counts over the total reads in that sample of DNA extracted from cells grown in the absence (control) or presence of aTc. The average fraction of pegRNA reads for the substitutions is shown as dots on the right Y-axis. Edits are observed up to 91 bp from the PAM site. Resulting means of four biological replicates are plotted. C Validation of the maximum editing distance through individually cloned pegRNAs. The mutations conferred by the pegRNAs all encode the G to C PAM mutation and a second mutation at varying distances. The 33, and 45 bps substitutions from the PAM site, confer a premature stop codon, whilst the pegRNAs encoding mutation either 72 or 91 bps from the PAM site resulted in a glycine to cysteine (72 bps) or isoleucine to asparagine (91 bps) substitution ( n = 21 each). The percentages indicate the fraction of clones that were phenotypically observed to be edited. Clones with an RLU/OD value below 3 × 10 3 were considered to be edited. The mutation at 91 nts (*) could only be confirmed by sequencing individual clones, as the amino acid change does not affect luciferase function. This showed an editing efficiency of 4.8 % (1 out of 21 tested colonies) with the correct genome edit.

Article Snippet: Chromosomal DNA from the pegRNA pool screening samples was extracted for Illumina library preparation using the Promega kit as described previously .

Techniques: Mutagenesis, Sequencing, Control, Biomarker Discovery, Clone Assay, Luciferase

Journal: Nature Communications

Article Title: Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

doi: 10.1038/s41467-025-59068-8

Figure Lengend Snippet: A Schematic overview of the principle of the split luc system. B Schematic overview of the pegRNA used to fuse a SmBit tag to pbp1A using mbPE. C PBP1a interacts with MpgA and RodZ. Each dot represents the average of 15 measurements of a technical replicate, with the size of the dot representing the SEM. Control strains only expressing pbp1A-SmBit (labeled as D39V) or pbp1A-SmBit together with the abundant DNA-binding protein HU-LgBit did not demonstrate bioluminescence. D PBP1a and MpgA co-localize in space and time. Live cell fluorescence microscopy of cells of strain VL7454 (mScarlet-PBP1a, GFP-MpgA) grown at 37 °C in C + Y medium. Scale bar: 2 mm. Demograph of exponentially growing cells of strains VL5532 (GFP-PBP1a) and VL5816 (GFP-MpgA). Representative data are shown from one biological replicate, data of at least 1,000 cells are plotted. E , F Sequential gene editing in S. pneumoniae using mbPE. Schematic overview of cloning regime with the two alternative pegRNA vector backbones (pVL4133, spec ; pVL7227, cat ) to generate a triple genome edited S. pneumoniae strain (Δ63- luc , Δ cps2A-stop , Δ lytA-stop ) in a sequential manner.

Article Snippet: Chromosomal DNA from the pegRNA pool screening samples was extracted for Illumina library preparation using the Promega kit as described previously .

Techniques: Control, Expressing, Labeling, Binding Assay, Fluorescence, Microscopy, Cloning, Plasmid Preparation