Development of a new genetic reference material system based on Saccharomyces cerevisiae cells

Abstract

As an important quality control link of molecular diagnosis, genetic reference materials (RMs) are widely used in various gene detection platforms such as mutation detection, gene quantification, and second generation sequencing. However, contamination, construction, and storage of existing genetic RMs still remain challenges. Here, we established a new genetic RM system based on Saccharomyces cerevisiae. We chose the non-small cell lung cancer (NSCLC) mutation hotspots in Kirsten rat sarcoma viral oncogene (KRAS) and epidermal growth factor receptor (EGFR), using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas9) system-mediated gene editing technology, combined with the high homologous recombination efficiency of Saccharomyces cerevisiae. A single copy of the target gene was inserted into the yeast genome, and the inserted target gene was stably inherited with the passage of yeast cells. The copy number calculation for the target gene can replays by cell counting. The RM system was evaluated by sequence, copy number, stability, and homogeneity. In summary, the recombinant yeast cell line has ease of construction and screening, stable genetic characteristics, accurate copy number calculation, and convenient culture and preservation. Our findings may provide new ideas and directions for the research and industrialization of genetic RMs.

Keywords: CRISPR-Cas9; EGFR; KRAS; Saccharomyces cerevisiae; genetic reference materials; homologous recombination.

Graphical abstract

Figure 1

Construction and optimization of recombinant S. cerevisiae genetic RMs (A) Three single guide RNAs (sgRNAs) were designed, and the results of 2SapI-Cas9-gRNA1, 2SapI-Cas9-gRNA2, and 2SapI-Cas9-gRNA3 plasmids were sequenced. (B) By transferring the single colonies grown on the plate to the new SD-Leu- plate, the number of colony growth was inversely proportional to the editing efficiency. (C) Schematic diagram of three homologous recombination templates. (D) Comparison of recombination efficiency of three homologous recombination templates.

Figure 2

Verification of calibration ability of recombinant yeast cell genetic RMs (A) The sequencing results of recombinant yeast genetic RMs. (B) Taking a recombinant yeast clone with KRAS mutation as an example, standard curves were drawn for the three pairs of primers, KRAS-Q1, KRAS-Q2, and ACT1, used in the experiment. (C) qPCR standard curve of recombinant template plasmid, human genomic DNA, and recombinant yeast genomic DNA. (D) The recombinant yeast cells were serially passaged, and the copy number of the target gene of 10th, 20th, 30th, 40th, 50th, and 60th generation yeast cells was detected by qPCR. (E) Verification of copy number stability of recombinant yeast cells stored in 20% glycerol at −80°C for 180 and 270 days.

Figure 3

Homogeneous treatment of recombinant yeast cells and human cells (A) The growth curve of Saccharomyces cerevisiae W303-1A strain. (B) The protoplast morphology of Saccharomyces cerevisiae W303-1A cells: (B-1) the yeast protoplast in the osmotic buffer (10 × 40 times magnification by optical microscopy); (B-2) with the addition of pure water to the yeast protoplast suspension, the cell swells; (B-3) protoplasts rose and broke after absorbing water for a period of time; and (B-4) the enzymatic hydrolysis time is too long and the protoplast cell atypicality increased.

Figure 4

Recombinant yeast cells mixed to mimic low-frequency mutations in tumor tissues (A) Representative images of DAPI nuclear staining of yeast protoplasts. (B) Detection of target mutation frequency of the samples by the next generation sequencing and micro-drop digital PCR.