Although this multicolor approach has expanded the potential of split-FP labeling, it has a bottleneck in multiplexing because of the limited number of available orthogonal split FP systems with different colors.
The GFP 11 and sfCherry 11 fragments allow simultaneous labeling of two different proteins. Recently, great advances have been made in split super-folder Cherry (sfCherry 1-10/11) as a second, orthogonal split FP system 2, 13, 14. Multiplexed visualization is tremendously beneficial for simultaneous comparisons of protein dynamics. However, labeling multiple proteins simultaneously in single cells has been challenging. Additionally, we have been able to generate a library of human cells with GFP 11-tagged endogenous proteins via CRISPR/Cas9-mediated homology-directed repair (HDR), and demonstrate that GFP 11-tag is compatible with a wide range of cellular proteins such as enzymes, receptors, transport proteins, and structural proteins 12. In particular, endogenously tagged cell lines can be produced by the efficient introduction of the short fragment (GFP 11) into a genomic locus without perturbing local genomic structure 2, 11. These self-complementing split GFP variants have already become a powerful and versatile tool for various imaging applications. These fragments retain the ability to bind to the identical GFP 11 fragment, so that reconstitution with GFP 11 produces a functional cyan or yellow FP. The majority of substitutions which lead to the spectral shifts in these variants are located within the large fragments (i.e., CFP 1-10 and YFP 1-10). By introducing additional substitutions into the GFP 1-10 fragment, cyan and yellow spectral variants were previously created and used to visualize localization patterns of cellular proteins 2, 10. The GFP 11 fragment has been used in numerous biological studies 3, 4: targeting nanomaterials in cells 5, 6, forming protein oligomeric structures 2, 7, verifying aggregation processes 8, and imaging protein localization in living cells 9. When expressed in the same cell, the GFP 1-10 D7 and GFP 11M3 OPT fragments (hereafter referred to as GFP 1-10/11) spontaneously interact with each other to form a functional GFP (Supplementary Fig. The short fragment, GFP 11M3 OPT, acts as an epitope tag when inserted into a gene of interest 2. In the self-complementing split GFP system, super-folder GFP is split between β-strands 10 and 11, rendering 214-amino acid and 16-amino acid fragments 1. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells the resulting images reveal nuclear localization of focal adhesion protein Zyxin. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP 1-10/11 pairs, offering multiplexed labeling of cellular proteins. We also circularly permutate GFP and synthesize the β-strand 7, 8, or 10 system. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP 1-10/11. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Self-complementing split fluorescent proteins (split FP 1-10/11) have become an important labeling tool in live-cell protein imaging.
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