清脆的
质粒
Cas9
生物
基因
分子生物学
遗传学
作者
Birthe Meineke,Simon J. Elsässer
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 169-180
被引量:1
标识
DOI:10.1007/978-1-0716-3251-2_12
摘要
Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.
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