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• W4387529447 abstract "ABSTRACT Targeted gene disruption is challenging in the dimorphic fungal pathogen Histoplasma due to the low frequency of homologous recombination. Transformed DNA is either integrated ectopically into the genome or maintained extrachromosomally by de novo addition of telomeric sequences. Based on a system developed in Blastomyces , we adapted a CRISPR/Cas9 system to facilitate targeted gene disruption in Histoplasma with 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 including RYP2 and SRE1 , where 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 endogenous Histoplasma regulatory sequence to drive guide RNA expression. These tools transform our ability to generate targeted mutations in Histoplasma . IMPORTANCE Histoplasma is 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 in Histoplasma . 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 in Histoplasma with high efficiency, thereby allowing disruption of multiple genes." @default.
• W4387529447 created "2023-10-12" @default.
• W4387529447 creator A5008617222 @default.
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• W4387529447 date "2023-10-11" @default.
• W4387529447 modified "2023-10-13" @default.
• W4387529447 title "Recyclable CRISPR/Cas9-mediated gene disruption and deletions in <i>Histoplasma</i>" @default.
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• W4387529447 doi "https://doi.org/10.1128/msphere.00370-23" @default.
• W4387529447 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/37819140" @default.
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