CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line, Allowing Rapid Knockdown of Protein Function

Abstract

Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles.The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.

A Chemical Genetics Approach for the Functional Assessment of Novel Cancer Genes

“…Using CRISPR-based targeting, however, we were able to engineer "homozygously" DD-tagged clones in a single round of transfection even in cell lines that were triploid or tetraploid for the locus of interest. Together with a recent study that developed a similar approach but was restricted to a single gene and a single cell line (38), these findings indicate that the Degron-KI approach should be broadly applicable to many genes and cell line models.…”

Affinity Proteomics to Study Endogenous Protein Complexes: Pointers, Pitfalls, Preferences and Perspectives

“…These resources have broadly facilitated implementation of affinity capture/MS strategies in mammalian tissue culture systems (66,68). Although CRISPR-based methods (69–72) are increasingly enabling tagging of endogenous proteins, numerous methods employ an additional copy of a tagged transgene. Three popular approaches for human transgene expression that we commonly use are the Flp-In T-Rex system (Life Technologies), BAC transgenesis, and episomal transfections (73,74).…”

Chemical Biology Strategies for Posttranslational Control of Protein Function

“…For example, it has now been demonstrated that destabilizing domains can be inserted upstream of exon 1 of Treacher Collins-Franceschetti syndrome1 (TCOF1) to conditionally regulate it by the addition of Shield-1 (Park et al, 2014). Because of the high efficiency of gene targeting, both alleles can be modified in a single cell leading to conditional regulation without interference from endogenous sources.…”