CRISPR-Cas9 mediated knock-in in mammalian cells for protein purification

Abstract

Protein purifications for pharmaceuticals have long been a very important position in medical and basic biochemistry studies. Protein production using E. coli is a simple process however, they cannot mimic protein modifications which many human proteins have for example, PTMs. Thus, recently, methods of producing and refining proteins by inducing protein over-expression within animal cells have been in the spotlight. In Chapter 1., this paper shows engineered mammalian cell lines for protein purification. CRISPR-Cas9 is powerful target-specific nuclease and transcriptional hotspot is a good safe harbor to insert protein expression sequence on the genome. HEK293E cells have a Tectorin expression vector at transcription of its genome. The protein expression vector is inserted by NHEJ-KI method on CRISPR-Cas9 mediated DNA double-strand break site. As such, if the process of produce proteins by inserting genes that can express target proteins into animal cells becomes common, it is expected that target proteins can be produced and refined more economically and easily. In Chapter 2., nuclease inhibition activity of anti-CRISPR made from the evolution process of virus to avoid CRISPR-Cas system which is immune system of prokaryotes is evaluated by targeted deep-sequencing. The use of anti-CRISPR as preventing non-target DNA from off-target activity of Cas9 is suggested.