Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system

Scialli, Nicoletta Schibeci Natoli; Colitti, Barbara; Bertolotti, Luigi; Pezzoni, Giulia; Martignani, Eugenio; Melega, Maverick; Brocchi, Emiliana; Rosati, Sergio

doi:10.1016/j.vetimm.2021.110286

Cited by 1 publication

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“…It is unclear why RNP1 failed to cut its target site in our test of the two-RNP strategy. The use of two gRNAs to edit a gene works in other systems [29][30][31][32], and it works on the C. reinhardtii chloroplast genome [33]. In two of these studies, the target cut sites were separated by 960 bp [31] and 32 to 98 bp [32]; for comparison, in our experiment the two target sites were separated by 767 bp, so the linear distance between the sites is unlikely to have hindered cutting by RNP1.…”

Section: One-rnp Strategy Vs Two-rnp Strategymentioning

confidence: 84%

Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis

2022

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Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.

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