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Modifying the potato tuber storage protein patatin targeting improved thermal stability.
Gene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals. Patatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations.
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A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants.
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Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica.
The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR-Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III-tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1'-tRNA(Gly) promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8-TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor plasmid resulted in markerless homologous recombination efficiency of over 64%. Homologous recombination was observed in 100% of transformants when nonhomologous end joining was disrupted. The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica.
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Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.
Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.
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Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA-ncRNA Expression Constructs.
CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.
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CRISPR fixes disease gene in viable human embryos.
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CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance.
Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.
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Application of CRISPR/Cas12i.3 for targeted mutagenesis in broomcorn millet (Panicum miliaceum L.).
A CRISPR/Cas12i.3-based gene editing platform is established in broomcorn millet (Panicum miliaceum) and used to create new elite germplasm for this ancient crop.
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Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.
CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.
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Genome editing in Penicillium funiculosum using in vitro assembled CRISPR-Cas9 ribonucleoprotein complexes.
The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in Penicillium funiculosum, which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with in vitro synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021).
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