We describe a rapid and highly efficient method to generate point mutations in Caenorhabditis elegans using direct injection of CRISPR-Cas9 ribonucleoproteins. This versatile method does not require sensitized genetic backgrounds or co-CRISPR selection-based methods, and represents a single strategy that can be used for creating genomic point mutations, regardless of location. As proof of principle, we show that knock-in mutants more faithfully report variant-associated phenotypes as compared to transgenic overexpression. Data for nine knock-in mutants across five genes are presented that demonstrate high editing efficiencies (60%), a reduced screening workload (24 F1 progeny), and a rapid timescale (4-5 d). This optimized method simplifies genome engineering and is readily adaptable to other model systems. KEYWORDS CRISPR Cas9 ribonucleoprotein Caenorhabditis elegans disease variantsNext-generation DNA sequencing technologies have enabled the rapid identification of clinical sequence variants, yet a significant gap still exists in characterizing their functional and pathological significance (Boyd et al. 2014). Moreover, allele frequency is often used as a surrogate to infer disease relevance without functional validation in animal models (Minikel and MacArthur 2016). Introducing site-specific variants in a rapid and facile manner in model organisms would greatly aid in unmasking the pathogenic potential of newly identified sequence variants of unknown significance. Recent technological advances such as the CRISPR-Cas9 genome editing system have revolutionized the ability to precisely engineer the genomes of the most prevalent model organisms used in biomedical research (Frokjaer-Jensen 2013;Doudna and Charpentier 2014;Sander and Joung 2014;Ma and Liu 2015;Dickinson and Goldstein 2016;Sugi 2016). Here, we have developed a simplified CRISPR-Cas9 genome editing method for generating point mutations in the model organism Caenorhabditis elegans. This simplified, optimized, and highly-efficient method obviates the need for sensitized genetic backgrounds, selection-based or co-CRISPR methods, and permits the generation of specific knock-in alleles into any strain background within 4-5 d.Several methods currently exist for engineering the C. elegans genome using CRISPR-Cas9, but the majority of these methods rely on specific genetic backgrounds or co-CRISPR strategies in which screening for the successful edit of one marker gene enriches for the genome edit of interest (Dickinson and Goldstein 2016). Although powerful, these methods do have limitations. For example, the dpy-10 co-CRISPR strategy introduces a point mutation that produces an easily observed dominant roller phenotype (Arribere et al. 2014). Although convenient, introducing selectable phenotype-bearing mutations is undesirable if the strain to edit or the desired point mutation of interest itself exhibits a similar phenotype or a phenotype that might be exacerbated or suppressed by the dpy-10 roller phenotype. Moreover, co-CRISPR strategies rely ...
BACKGROUND AND PURPOSEIonotropic GABA receptors are evolutionarily conserved proteins that mediate cellular and network inhibition in both vertebrates and invertebrates. A unique class of excitatory GABA receptors has been identified in several nematode species. Despite wellcharacterized functions in Caenorhabditis elegans, little is known about the pharmacology of the excitatory GABA receptors EXP-1 and LGC-35. Using a panel of compounds that differentially activate and modulate ionotropic GABA receptors, we investigated the agonist binding site and allosteric modulation of EXP-1 and LGC-35. EXPERIMENTAL APPROACHWe used two-electrode voltage clamp recordings to characterize the pharmacological profile of EXP-1 and LGC-35 receptors expressed in Xenopus laevis oocytes. KEY RESULTSThe pharmacology of EXP-1 and LGC-35 is different from that of GABA A and GABA A -ρ receptors. Both nematode receptors are resistant to the competitive orthosteric antagonist bicuculline and to classical ionotropic receptor pore blockers. The GABA A -ρ specific antagonist, TPMPA, was the only compound tested that potently inhibited EXP-1 and LGC-35. Neurosteroids have minimal effects on GABA-induced currents, but ethanol selectively potentiates LGC-35. CONCLUSIONS AND IMPLICATIONSThe pharmacological properties of EXP-1 and LGC-35 more closely resemble the ionotropic GABA A -ρ family. However, EXP-1 and LGC-35 exhibit a unique profile that differs from vertebrate GABA A and GABA A -ρ receptors, insect GABA receptors and nematode GABA receptors. As a pair, EXP-1 and LGC-35 may be utilized to further understand the differential molecular mechanisms of agonist, antagonist and allosteric modulation at ionotropic GABA receptors and may aid in the design of new and more specific anthelmintics that target GABA neurotransmission. AbbreviationsEXP-1, expulsion defective; LGC-35, ligand-gated ion channel
MRSA is a major pathogen worldwide and its infections are associated with increased morbidity and mortality, in comparison with other S. aureus infections. The study was designed to characterize the mecA gene and staphylococcal cassette chromosome (SCCmec) in methicillin resistance S. aureus (MRSA). A total of 20 presumptive S. aureus strains were collected from one of the teaching hospitals in Selangor. Using standard biochemical tests, all the isolates were verified as S. aureus. When tested against cefoxitin, 80% of the isolates were confirmed as MRSA. All the MRSA isolates were further subjected to polymerase chain reaction (PCR) to detect the presence of mecA gene. Nine out of the 16 MRSA isolates (56%) were mecA positive, whereas the remaining four were mecA negative. The 16 MRSA with positive mecA gene were further subjected to SCCmec typing of type I, II, III, IV and V. The most frequent SCCmec types were type III (56%) followed by type II (33%), and type IV (11%). None of the isolates were of SCCmec type I or V. Our study indicates that SCCmec type III is predominant among the isolates which is in agreement with other studies conducted on clinical strains of MRSA.
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