MethodsX (Jan 2021)
A cost efficient protocol to introduce epitope tags by CRISPR-Cas9 mediated gene knock-in with asymmetric semi-double stranded template
Abstract
To study the biological function of uncharacterized proteins, specific antibodies are in high demand. However, the production of desirable antibodies such as highly specific or high affinity is not always successful. Furthermore, even if commercially available antibodies exist, the cost, quality, and accessibility often differ from country to country. In comparison, epitope tags are reliable and economical options since good antibodies against major epitope tags are commercially available. Although exogenously expressed epitope-tagged protein appears as a timely method, the excessive protein production may not faithfully recapitulate its biology. Thanks to the recent advances in genome editing by CRISPR-Cas9, HDR-mediated endogenous protein tagging has become an accessible approach for many labs. However, currently the synthesis of long (>100 bp), chemically modified oligos can be time-consuming and costly. To develop a reliable, simple, and cost-effective epitope-tagging method that requires minimal materials and apparatus, we focus on an approach utilizing two non-chemically modified shorter-annealed oligos (semi-dsODNs) mediated HDR for epitope tags insertion. We also use a cationic lipid chemical, polyethyleneimine (PEI), for plasmid delivery to minimize the cost and materials used while a considerable success rate could be achieved.• This protocol provides a more economical way to generate CRISPR-Cas9 mediated gene knock-in.• This protocol provides a simplified design of semi-dsODN without chemical modification on the oligos.• This protocol provides a simplified experimental procedure. In vitro assembled Cas9 complex and electroporation are not required.
