an-in-vitro-assay-to-detect-trna-isopentenyl-transferase-activity

An <em>In Vitro</em> Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

crispr-mediated-genome-editing-human-fungal-pathogen-candida

CRISPR-mediated Genome Editing of the Human Fungal Pathogen <em>Candida albicans</em>

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Ball State University

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in , , , and  These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both  and  rely heavily on homology-directed repair, whereas  and  use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens  and , as well as in the related yeasts  and  The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the  Zn-binding domain, a known regulator of  filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Douglas A. Bernstein

Department of Biology

Ball State University

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Douglas A. Bernstein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Ball State University

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Department of Biology