Inducing the CRISPR-Cas9 expression in solid medium
For continuous induction on solid medium, transformants carrying the
Cas9 plasmid are grown overnight in liquid SC-Rep medium. The optical
density at 600nm of the culture is determined, and aliquots are plated
on SC-Ind plates as well as on repressive SC-Rep medium. Colonies are
grown, counted and plates are compared in order to estimate the survival
rate (number of colonies obtained on induction medium divided by number
of colonies obtained on repression medium, expressed as a percentage).
Induction plates are replicated on YDP-Rep, grown and examined for
colony color; both sectored and completely red colonies being scored
together. The rate of red colonies is expressed as a percentage of the
total number of colonies. In experiments that were used to compute the
survival and ade- rates, the minimum number of colonies counted
on SC-Rep is 50, and usually around 500. All experiments were performed
on at least two independent transformants and repeated at least twice
per transformant. Controls without the gRNA (transformation with pCFYF)
and controls without plasmid (with uracil added to media) were prepared
and analyzed in the same way. Standard deviations are calculated and
shown as error bars in graphs.