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Targeted genome editing in Caenorhabditis elegans using CRISPR /Cas9

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Utilization of programmable nucleases to generate DNA lesions at precise endogenous sequences has transformed the ability to edit genomes from microbes to plants and animals. This is especially true in organisms that previously lacked the means to engineer precise genomic changes, like Caenorhabditis elegans. C. elegans is a 1 mm long free‐living, nonparasitic, nematode worm, which is easily cultivated in a laboratory. Its detailed genetic map and relatively compact genome (~100 megabases) helped make it the first metazoan to have its entire genome sequenced. With detailed sequence information came development of numerous molecular tools to dissect gene function. Initially absent from this toolbox, however, were methods to make precise edits at chosen endogenous loci. Adapting site‐specific nucleases for use in C. elegans, revolutionized studies of C. elegans biology. Zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), and then CRISPR‐associated protein 9 (Cas9) were used to target specific endogenous DNA sequences to make double‐strand DNA breaks (DSBs). Precise changes could be engineered by providing repair templates targeting the DSB in trans. The ease of programming Cas9 to bind and cleave DNA sequences with few limitations has led to its widespread use in C. elegans research and sped the development of strategies to facilitate mutant recovery. Numerous innovative CRISPR/Cas9 methodologies are now primed for use in C. elegans.

(a) Diagram of Cas9 bound to a single guide RNA and its DNA target site. Guide RNA sequences underlined in (b)–(d), target the same DNA site as shown in (a). (b) Dual guide RNA consisting of a crRNA and separate tracrRNA. (c) Diagram of a single guide RNA made from concatenating the crRNA and tracrRNA. (d) Diagram of the modified singe guide RNA(F+E). The A‐U base flip and RNA loop extension are highlighted in yellow.
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Cas9 can be repurposed as a platform to recruit effector proteins to visualize nucleic acid dynamics or modulate transcription. Mutations in Cas9's nuclease domains inactivate its endonucleolytic activity while preserving its DNA binding activity. (a) Categories of effector proteins. (b) The effectors have been directly tethered to dCas9, (c) dCas9 has been fused to a SunTag that can binds single chain variable fragment (scFv) antibodies fused to effectors, or (d) the guide RNA has be fused to RNA sequences, such as MS2 repeats, that recruit proteins (MCP) fused to effector proteins. Tethering multiple effectors allows for amplification of fluorescence or more potent transcriptional activation or repression. (d) Cas9 can also be tiled across DNA targets to recruit more effectors and achieve greater amplification.
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Efficient methods to screen or select for Cas9‐edited C. elegans. (a) A DNA transformation screen is shown. P0 worms are injected with DNA encoding Cas9, guide RNAs, repair templates, and an easily scored transformation marker. Offspring of injected animals are picked based on expression of DNA transformation markers (red) and scored for the presence of Cas9‐directed edits. Worms homozygous for the mutation of interest are then chosen. (b) A Co‐CRISPR/Co‐conversion screen is shown. Worms are microinjected with DNA repair templates and Cas9 and guides are supplied by mRNA, DNA, or RNPs microinjection. Offspring of injected animals are screened based on editing at a reference locus, in this example, dpy‐10. When appropriately edited, dpy‐10(gof) yields a roller phenotype. Rol worms are then scored for the presence of an edit at the second locus and homozygous animals picked. Selection away from Rol, returns dyp‐10(+) for rapid recursive mutagenesis. (c) An example of a selection‐based mutant strategy using a self‐excising repair cassette. P0s are microinjected with a DNA repair template while Cas9 and guide RNAs are provided by mRNAs, DNA, or RNPs injection. Appropriate cleavage and repair using the DNA repair template, yields hygromycin resistant worms that are rollers, due to sqt‐1(gof). Due to engineered stop codons in the cassette, the insertion yields a loss‐of‐function allele for the gene of interest. Homozygous hygromycin resistant, Rol worms are picked and heat‐shocked to induce Cre expression. Cre recombines the two loxP sites excising the cassette and leaving a small genetic scar, and gfp in frame with the gene of interest.
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