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Third, recovery of target genes after screening and sorting is typically done from large pools (10 3–10 7) of collected cells with subsequent random picking of just a few (10–200) clones from the pool that significantly reduces the chance of finding the best variant. Higher mutation rates can be achieved with CRISPR/Cas9 editing technology 28 or via in vitro mutagenesis, 27 but comes with a limited library size of 10 5–10 6 independent clones. Second, in situ diversification of target genes using a cytidine deaminase 25, 26, 29 or viral replication 30 has a low mutation rate of only 1–3 mutations per kilobase pair in comparison to 9–16 mutations per kilobase pair for regular error-prone PCR typically used for FP development. 26- 28 However, both commonly used single gene copy delivery methods, such as electroporation and retroviral transduction, and establishing stable cell lines, are time-consuming and laborious, and complicated by apoptosis, low efficiency of stable gene integration, and long cell doubling time. First, all previously developed methods for directed molecular evolution in cultured cells from vertebrates involve establishing cell lines stably maintaining target genes, in a way where any given cell expresses ideally no more than one copy of a target gene. 26- 28 However, the proposed methods did not find wide adaptation among protein engineers due to several limitations and drawbacks. Indeed, several studies demonstrated the possibility to evolve FPs in cultured chicken 25 or mammalian cells. 24 In this regard, vertebrate cell lines represent a promising expression host for directed molecular evolution of FPs. 22, 23 Yeasts, although eukaryotic cells, which provide convenience of large gene library expression like bacteria, may not serve as an ideal host system for FPs as it was shown that brightness in yeast cells is not necessarily retained in mammalian cells. 21 Ideally, the development of functional proteins for in vivo imaging should be performed in an environment physiologically relevant to the final hosts to ensure proper protein folding, localization, and posttranslational modification. 16- 18 This phenomenon is particularly well documented for bacteriophytochrome-based FPs, 16, 19, 20 whose fluorescence relies on the incorporation of the chromophore biliverdin (BV) from the bulk. 13- 15 However, high molecular brightness, commonly screened for in bacterial cells, does not always correspond to high intracellular brightness when expressed in cultured mammalian cells 8, 9, 16 or in vivo. 13, 14 FPs are usually optimized via directed molecular evolution by iteratively generating and screening large gene libraries in bacterial cells. 8- 12 Among all biochemical characteristics, intracellular brightness is one of the most crucial properties for in vivo applications that protein engineers and developers choose to optimize before everything else. 7 However, all naturally occurring chromoproteins have to be modified, optimized, or even reengineered in order to be utilized for fluorescence imaging in vivo. 1 Since the cloning of the first green FP from jellyfish Aequorea victoria in 1992, 2 a myriad of chromoproteins with various spectral and biochemical properties have been cloned from diverse natural sources such as corals, 3 fish, 4 plants, 5 soil bacteria, 6 and cyanobacteria. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.įluorescent proteins (FPs) became indispensable tools for in vivo imaging of cellular and subcellular structures in model organisms. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging.

The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10 7 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness.

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