Fig. 1

CRISPR-induced mutation frequency in CAN1 and plasmid loss assays. a Frequency of mutations in the CAN1 gene assessed by the formation of colonies on plates containing canavanine, which is toxic to CAN1⁺ yeast. The plasmids in strain PT141 are specified on the x-axis, and each column is a single experiment. Frequencies were calculated from the number of colonies on canavanine-containing plates compared with no drug (the media lacked uracil and leucine to select for both plasmids and also arginine to allow canavanine toxicity). b The rates of plasmid loss were measured, since the endonuclease complex is encoded on two separate plasmids: pCas9 and pCAN1-guide. Yeast cells (TEF1-GFP from the GFP collection) containing both plasmids were grown overnight with selection for both plasmids and then 500 cells were plated on medium that selects for the pCas9 plasmid (−leucine), the pCAN1-guide plasmid (−uracil) or both (−lecuine, −uracil). The resulting colonies were compared with growth without selection. The overnight growth medium contained either glucose (blue bars) or galactose (orange bars), the latter medium induces expression of the Cas9 gene. At least one of the plasmids, pCas9 or pCAN1-guide, was lost from 10 to 40% of cells pre-grown glucose medium. This loss rate increased to nearly 100% of cells, when the Cas9 gene was induced with galactose. The pCAN1-guide plasmid is lost more readily than the pCas9 plasmid. To determine if this loss rate was caused by the endonuclease activity, we repeated the experiment with a dead version of Cas9, which contains D10A and H840A substitutions. The plasmid loss rate on galactose was much less (30–50%) with an inactive Cas9 compared with the active Cas9, indicating that plasmid loss is associated with endonuclease function. Error bars represent exact binomial 95% confidence intervals. c The number of colonies observed on YPD (blue bars), −ARG (light green bars) and -ARG NAT (dark green bars, this media selects for the plasmid) media following galactose induction of a single plasmid encoding expression of both the Cas9 and guide targeting CAN1, and two control plasmids. Five hundred cells were plated and each column is a single experiment. d The frequency of mutations in the CAN1 gene were assessed by comparing the formation of colonies on canavanine plates, with those containing no drug. The plasmids present in strain PT141 are specified on the x-axis, and each column is a single experiment. Frequencies were calculated both without selection for the plasmid (light green bars) and with NAT selection (dark green bars). e The effect upon viability of the different endonuclease plasmids was assessed by counting the viability of cells. One thousand five hundred HTB2-GFP cells (from the GFP library) that had been transformed with the plasmids stated on the x-axis were plated onto glucose (dark green bars, CAS9 expression OFF) and galactose media (orange bars, CAS9 expression ON), in 3 replicates (4500 cells in total). Bars represent mean and error bars represent standard deviation. **, P < 0.01, *, P < 0.05, n.s., non-significant; Welch’s two-sample t-test performed on 3 replicates