Recyclable CRISPR/Cas9 mediated gene disruption and deletions inHistoplasma

Abstract

AbstractTargeted gene disruption is challenging in the dimorphic fungal pathogenHistoplasmadue to the low frequency of homologous recombination. Transformed DNA is either integrated ectopically into the genome or maintained extra chromosomally byde novoaddition of telomeric sequences. Based on a system developed inBlastomyces, we adapted a CRISPR/Cas9 system to facilitate targeted gene disruption inHistoplasmawith high efficiency. We express a codon-optimized version of Cas9 as well as guide RNAs from a single ectopic vector carrying a selectable marker. Once the desired mutation is verified, one can screen for isolates that have lost the Cas9 vector by simply removing the selective pressure. Multiple mutations can then be generated in the same strain by retransforming the Cas9 vector carrying different guides. We used this system to disrupt a number of target genes includingRYP2andSRE1where loss-of-function mutations could be monitored visually by colony morphology or color, respectively. Interestingly, expression of two guide RNAs targeting the 5’ and 3’ ends of a gene allowed isolation of deletion mutants where the sequence between the guide RNAs was removed from the genome. Whole-genome sequencing showed that the frequency of off-target mutations associated with the Cas9 nuclease was negligible. Finally, we increased the frequency of gene disruption by using an endogenousHistoplasmaregulatory sequence to drive guide RNA expression. These tools transform our ability to generate targeted mutations inHistoplasma.ImportanceHistoplasmais a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events inHistoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions inHistoplasmawith high efficiency, thereby allowing disruption of multiple genes.