Dimorphic fungi in the genera
Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn
- NSF-PAR ID:
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Microbiology
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
Dimorphic fungi in the genera
Blastomyces, Histoplasma, Coccidioides, and Paracoccidioidesare important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA‐guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidisin particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9‐expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomycesusing Agrobacterium‐mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene‐editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium‐mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomycesfrozen stocks
Nanobodies (nAbs) are recombinant antigen‐binding variable domain fragments obtained from heavy‐chain‐only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well‐established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC.
This article was corrected on 26 June 2021. See the end of the full text for details.
Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First‐strand cDNA synthesis; VHH and VHrepertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small‐scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb‐pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. colibacterial strains
CRISPR‐Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large‐scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA‐programmable nature of CRISPR‐Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR‐suppressor scanning. In a typical CRISPR‐suppressor scanning experiment, a pooled library of single‐guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ. This cellular pool of protein variants is then challenged with drug treatment to identify mutations conferring a fitness advantage. Drug‐resistance mutations identified with this approach can not only elucidate drug mechanism of action but also reveal deeper mechanistic insights into protein structure‐function relationships. In this article, we outline the framework for a standard CRISPR‐suppressor scanning experiment. Specifically, we provide instructions for the design and construction of a pooled sgRNA library, execution of a CRISPR‐suppressor scanning screen, and basic computational analysis of the resulting data. © 2022 Wiley Periodicals LLC.
Basic Protocol 1: Design and generation of a pooled sgRNA library Support Protocol 1: sgRNA library design using command‐line CRISPOR Support Protocol 2: Production and titering of pooled sgRNA library lentivirus Basic Protocol 2: Execution and analysis of a CRISPR‐suppressor scanning experiment
Solid‐phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non‐chromatographic procedure that will enable routine solid‐phase synthesis and purification of long RNA strands. The optimized five‐step process is based on bio‐orthogonal inverse electron demand Diels‐Alder chemistry between
trans‐cyclooctene (TCO) and tetrazine (Tz), and entails solid‐phase synthesis of RNA on a photo‐labile support. The target oligonucleotide strands are selectively tagged with Tz while on‐support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76‐nt long tRNA and 101‐nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR‐Cas9 experiments. © 2021 Wiley Periodicals LLC. Basic Protocol: Five‐step non‐chromatographic purification of synthetic RNA oligonucleotides Support Protocol 1: Synthesis of the components that are required for the non‐chromatographic purification of long RNA oligonucleotides. Support Protocol 2: Solid‐phase RNA synthesis
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein)‐mediated genome editing has revolutionized fundamental research and plant breeding. Beyond gene editing, CRISPR/Cas systems have been repurposed as a platform for programmable transcriptional regulation. Catalytically inactive Cas variants (dCas), when fused with transcriptional activation domains, allow for specific activation of any target gene in the genome without inducing DNA double‐strand breaks. CRISPR activation enables simultaneous activation of multiple genes, holding great promise in the identification of gene regulatory networks and rewiring of metabolic pathways. Here, we describe a simple protocol for constructing a dCas9‐mediated multiplexed gene activation system based on the CRISPR‐Act3.0 system. The resulting vectors are tested in rice protoplasts. © 2022 Wiley Periodicals LLC.
Basic Protocol 1: sgRNA design and construction of CRISPR‐Act3.0 vectors for multiplexed gene activation Basic Protocol 2: Determining the activation efficiency of CRISPR‐Act3.0 vectors using rice protoplasts