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

Abstract

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 ...

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit results in the creation of two genes

“…52 ontology analysis suggests that cuticle development and innate immune responses are regulated by nurf-23 1 ( Table S2) consistent with the role of its orthologs in regulating immunity and melanocyte proliferation in nurf-1 produces multiple transcripts encoding multiple protein isoforms 28 Our results suggest that selection acted repeatedly on C. elegans nurf-1 during laboratory growth. The 29 molecular nature of NURF-1, an essential subunit of the NURF chromatin remodeling complex, is 30 surprising for a hotspot gene.…”

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

“…However, we have not found a strict correlation between positives and plates with a high number of dpy-10 edited animals ( Figure S1). Alternatively, other co-injection markers as extrachromosomal plasmids harboring fluorescent reporters, antibiotic resistance genes, or dominant alleles can be used (Norris et al 2015;Prior et al 2017;). However, the use of plasmids could be a source of variability (mutations, distinct DNA preparations, etc.…”

CRISPR-Cas9 human gene replacement and phenomic characterization inCaenorhabditis elegansto understand the functional conservation of human genes and decipher variants of uncertain significance

“…8). To date, the most widely used genome editing-based human disease variant 318 assessment strategy in C. elegans uses sequence alignments to identify and engineer the 319 corresponding amino acid change into the orthologous C. elegans gene (Bend et al, 320 2016;Bulger et al, 2017;Canning et al, 2018;Pierce et al, 2018;Prior et al, 2017;321 Sorkaç et al, 2016;Troulinaki et al, 2018). A major advantage of this approach is that by 322 using the C. elegans gene, intronic regulation, protein-protein interactions, subcellular 323 localization, and biochemical activity of the protein of interest are, by design, perfectly 324 modeled by C. elegans.…”

“…This presents an important consideration when 328 attempting to replace C. elegans proteins that must interact in extremely precise 329 heteromeric complexes to perform their molecular functions (e.g. certain ion channel 330 subunits) (Bend et al, 2016;Prior et al, 2017). The most obvious limitation of this 331 approach is that it can only be used to study orthologous amino acids.…”