Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans.

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.

Pharmacological characterization of the excitatory 'Cys-loop' GABA receptor family in Caenorhabditis elegans.

BACKGROUND AND PURPOSE: Ionotropic 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 well-characterized 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 APPROACH: We used two-electrode voltage clamp recordings to characterize the pharmacological profile of EXP-1 and LGC-35 receptors expressed in Xenopus laevis oocytes. KEY RESULTS: The pharmacology of EXP-1 and LGC-35 is different from that of GABAA and GABAA -ρ receptors. Both nematode receptors are resistant to the competitive orthosteric antagonist bicuculline and to classical ionotropic receptor pore blockers. The GABAA -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 IMPLICATIONS: The pharmacological properties of EXP-1 and LGC-35 more closely resemble the ionotropic GABAA -ρ family. However, EXP-1 and LGC-35 exhibit a unique profile that differs from vertebrate GABAA and GABAA -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.