Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Species.

Many species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all species. We describe here an adaption of a previously developed CRISPR system in that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains and a selectable nourseothricin marker. It can be used for markerless editing in , , and We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in We show that editing can easily be carried out in multiple isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in and species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with is particularly common in South America and Asia, and infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the species group, including the sister species and We have also constructed a separate system for gene editing in .