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A Quantitative Comparison of the Knockdown Efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 in Caenorhabditis elegans
Author(s) -
Patel Anya,
Agrawal Nikki,
Feldman Sydney,
Hauser Paul,
De Luke
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.0r414
Subject(s) - crispr , cas9 , caenorhabditis elegans , gene knockdown , biology , endonuclease , green fluorescent protein , plasmid , gene , crispr interference , genetics , computational biology
Currently, many gene editing studies employ CRISPR/Cas9, however its low accuracy and knockdown efficiency is problematic. CRISPR‐Cas12, a newer and potentially more accurate variant, is hypothesized to have a higher knockdown efficiency than CRISPR/Cas9 in Saccharomyces cerevisiae and various plants. However this has not been quantitatively verified in mammalian cells. Our study compares the loss of gene function efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 through decreasing green fluorescent protein (GFP) expression in Caenorhabditis elegans via CRISPR‐mediated deletion. We used the LP162 strain of C. elegans which endogenously expresses GFP. We used C. elegans due to the similarity of molecular pathways to those of humans, their simple anatomy, and their fast reproductive cycle. Through bacterial transformation, we expressed our modified plasmid (containing the gRNA sequence, Cas endonuclease sequence, and C. elegans ‐specific promoters) in Escherichia coli . The C. elegans subsequently ingested the transformed bacteria. Fluorescence and protein expression were used as assays to determine the efficacy of CRISPR‐mediated GFP deletion. We hope to show a quantitative assessment of the difference in loss of function efficiencies between CRISPR/Cas9 and CRISPR‐Cas12 when used to delete GFP in C. elegans . Our goal is for these findings to be valuable for researchers seeking to use the most efficient Cas endonuclease for gene knockdown experiments.