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 ...
Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser and Thr in rat Ric-8A (corresponding to Ser and Thr in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser and Thr are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser and Ser, were also phosphorylated. Mutation of the genomic copy of in to encode alanine in the homologous sites resulted in characteristic reduction-of-function phenotypes that are associated with defective G and G signaling, including reduced locomotion and defective egg laying. The phosphorylation site mutant phenotypes were partially rescued by chemical stimulation of G signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.