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Highly improved cloning efficiency for plasmid-based CRISPR knock-in in C. elegans.

PMID: 34816097 (view PubMed database entry)
DOI: 10.17912/micropub.biology.000499 (read at publisher's website )
PMCID: PMC8606032 (free full text version available)

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Ella DeMott, Daniel J Dickinson, Ryan Doonan,

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in <i>C. elegans</i> (Dickinson <i>et al</i>. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin <i>et al</i>. 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.

MicroPubl Biol (microPublication biology)
[2021, 2021:]

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