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[
STAR Protoc,
2021]
In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C.elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C.elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
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Watts JK, Xu P, Ozturk AR, Gallant J, Dokshin GA, Mir A, Idrizi F, Krishnamurthy PM, Shin M, Lawson ND, Ghanta KS, Liu P, Yoon Y, Gneid H, Edraki A, Chen Z, Rivera-Perez JA, Sontheimer E, Mello CC, Zhang XO
[
Elife,
2021]
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (<i>Caenorhabditis elegans</i>, zebrafish, mice) and in cultured human cells.
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[
Genetics,
2020]
CRISPR genome editing has revolutionized genetics in many organisms. In the nematode <i>Caenorhabditis elegans</i> one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make <i>C. elegans</i> by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.
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Rettmann, Aubrie, Waterland, Skye, Huang, George, DeMott, Ella, Sylvester, Melynda, Dickinson, Daniel J, Rhodes, Anita, Flynn, Abbey, Alicea, Persephone, Blanco, Sara, Ren, Cassie, Koh, Alex, Doonan, Ryan, de Jesus, Bailey, Meng, Carrie
[
MicroPubl Biol,
2021]
The self-excising cassette (SEC) knock-in approach uses hygromycin selection and a visible roller phenotype to identify knock-ins, followed by a heat-shock induced excision of these visible markers to yield a seamless insertion of a fluorescent protein into the genome (Dickinson et al. 2015). Compared to protocols that utilize Cas9 protein and linear DNA repair templates (Paix et al. 2015; Dokshin et al. 2018; Ghanta and Mello 2020), the plasmid-based SEC approach employs a simpler screening strategy but requires more worms to be injected (Dickinson and Goldstein 2016).
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[
MicroPubl Biol,
2021]
Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.