Huang, George et al. (2021) MicroPubl Biol

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).

Ghanta KS et al. (2021) STAR Protoc "Microinjection for precision genome editing in Caenorhabditis elegans."

Mello CC, Ghanta KS, Ishidate T

[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).

DeMott, Ella et al. (2021) MicroPubl Biol

Doonan, Ryan, DeMott, Ella, Dickinson, Daniel J

[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.