scholarly journals Use of RNA-Protein Complexes for Genome Editing in Non-albicans Candida Species

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nora Grahl ◽  
Elora G. Demers ◽  
Alex W. Crocker ◽  
Deborah A. Hogan
Keyword(s):  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Promoter Activity ◽  
Protein Complexes ◽  
Content Type ◽  
Guide Rna ◽  
Genome Modification ◽  
Cas9 Protein ◽  
Rna Protein Complexes

ABSTRACT Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.

scholarly journals Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Kyunghun Min ◽  
Yuichi Ichikawa ◽  
Carol A. Woolford ◽  
Aaron P. Mitchell
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Drug Targets ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Content Type ◽  
Biological Features ◽  
Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

scholarly journals Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Henry Ng ◽  
Neta Dean
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Virulence Factors ◽  
Fungal Pathogen ◽  
Fungal Virulence ◽  
Important Conclusion ◽  
Content Type ◽  
Guide Rna ◽  
Improve Efficiency ◽  
Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

scholarly journals CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance inAspergillus fumigatus

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Takashi Umeyama ◽  
Yuta Hayashi ◽  
Hisaki Shimosaka ◽  
Tatsuya Inukai ◽  
Satoshi Yamagoe ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Genome Editing ◽  
Genetic Recombination ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Content Type ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Increased Susceptibility

ABSTRACTA pan-azole-resistantAspergillus fumigatusstrain with thecyp51Amutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containingcyp51Awith the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

scholarly journals Establishing CRISPR/Cas9 in Lipomyces starkeyi

2019 ◽  
Vol 2 (2) ◽  
pp. 51-52
Author(s):  
Zoe Lau ◽  
David Stuart ◽  
Bonnie Mcneil
Keyword(s):  
Protein Expression ◽  
Genome Editing ◽  
Oleaginous Yeast ◽  
Lipomyces Starkeyi ◽  
Guide Rna ◽  
Intracellular Lipids ◽  
Dry Cell Weight ◽  
Cas9 Protein ◽  
High Concentrations ◽  
Dry Cell

The goal of this project was to adapt the Yarrowia lipolytica plasmid based CRISPR/Cas9 system for usage in Lipomyces starkeyi. Lipomyces starkeyi is an oleaginous yeast, which synthesizes and stores high amounts of intracellular lipids. This specific yeast can store lipids at concentrations higher than 60% of its dry cell weight. Due to these high concentrations of lipids, L. starkeyi is a desired organism for the production of biofuels and other oleochemicals. However, there is a lack of knowledge and of genetic tools when trying to engineer the cells to produce these lipids for our use. The genome editing tool, CRISPR/Cas9 is efficient and simple, therefore desirable for the engineering of L. starkeyi. The goal was achieved by replacing the Y. lipolytica promoter with a L. starkeyi promoter, inserting guide RNA, as well as confirming cas9 protein expression.

scholarly journals Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

2018 ◽  
Vol 115 (38) ◽  
pp. 9351-9358 ◽  
Author(s):  
Robert M. Yarrington ◽  
Surbhi Verma ◽  
Shaina Schwartz ◽  
Jonathan K. Trautman ◽  
Dana Carroll
Keyword(s):  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Yeast Genome ◽  
Target Sequence ◽  
Practical Applications ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Wide Range

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.

scholarly journals A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Lauren Wensing ◽  
Jehoshua Sharma ◽  
Deeva Uthayakumar ◽  
Yannic Proteau ◽  
Alejandro Chavez ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Human Disease ◽  
Transcriptional Repression ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Fungal Infections ◽  
Content Type ◽  
Guide Rna ◽  
Crispr Interference ◽  
Functional Genomic Analysis

ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.

scholarly journals A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Milan Gerovac ◽  
Laura Wicke ◽  
Kotaro Chihara ◽  
Cornelius Schneider ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Rna Binding ◽  
Protein Complexes ◽  
Noncoding Rnas ◽  
Phage Infection ◽  
Complex Data ◽  
Human Pathogen ◽  
Content Type ◽  
Biological Stress ◽  
Rna Protein Complexes

ABSTRACT The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

scholarly journals Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing

2020 ◽  
Vol 26 ◽  
pp. 176-182
Author(s):  
I. S. Hnatiuk ◽  
O. I. Varchenko ◽  
M. F. Parii ◽  
Yu. V. Symonenko
Keyword(s):  
Genetic Transformation ◽  
Genome Editing ◽  
Plant Genome ◽  
Bacterial Protein ◽  
Rna Sequences ◽  
Genetic Construct ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Genetic Constructs

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods. Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression. 

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