Proteomics in the genome engineering era

Abstract

Genome engineering experiments used to be lengthy, inefficient, and often expensive, preventing a widespread adoption of such experiments for the full assessment of endogenous protein functions. With the revolutionary clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, genome engineering became accessible to the broad life sciences community and is now implemented in several research areas. One particular field that can benefit significantly from this evolution is proteomics where a substantial impact on experimental design and general proteome biology can be expected. In this review, we describe the main applications of genome engineering in proteomics, including the use of engineered disease models and endogenous epitope tagging. In addition, we provide an overview on current literature and highlight important considerations when launching genome engineering technologies in proteomics workflows.

Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry

“…With advances in the field of genome engineering, it has become feasible and efficient to introduce epitope tags on endogenous proteins. 40 To explore iMixPro in the context of tagged endogenous proteins, we introduced a 3xFLAG tag on the N-terminus of the JIP3 protein. The cell line was derived from the HCT116 colon carcinoma cell line and generated by rAav-based homologous recombination.…”

An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

“…This will allow a side-by-side comparison between classical Co-IP pull down experiments and binary approaches such as yeast two-hybrid and MAPPIT4445. While most of the published Co-IP data involves forced expression of bait and prey proteins, the revolution of genome engineering tools now allows the rapid engineering of epitope tags in endogenous proteins as demonstrated in this work37. Further optimization and streamlining of the SRM protocol for detection of the PQS peptides may allow the assessment of small molecule effects on protein expression or on PPIs as shown for the nutlin3 effect on the p53 - MDM2 interaction.…”

“…This can cause artifacts as the PQS tag may perturb protein folding, or have unexpected effects on transcription or translation. We focused all genome engineering efforts on the C-terminus of the native protein, as we rationalized the risk of interference with regulatory elements is lower at this end of the protein3637. Nevertheless, at this point it is hard to predict interference effects of fused sequences on protein expression, folding or interactions and use of tagging by genome editing should be approached empirically.…”

Multiplexed imaging of intracellular protein networks

“…Developing further orthogonal approaches for live cell labeling of tagged proteins with small, bright and photostable fluorophores with narrow emission spectra is a promising path to improve multiplexed imaging in live cells. The recent CRISPR/Cas9 technology enables endogenous tagging of the target proteins, thereby considerably strengthens this direction . Further developments of infrared fluorophores for live cell imaging are plausible to take place, thereby further enlarging the usable part of the spectra and increase the number of components that could be co‐imaged.…”