Intracellular Intein-Based Protein Editing: A Breakthrough in Precision Modification of Endogenous Proteins

Studying proteins in their native environment is essential for understanding cellular biology, but conventional labeling methods, such as fluorescent tags or antibodies, are often disruptive. These approaches can interfere with protein function, lack temporal precision, and are generally limited to genetically encodable tags. Small-molecule labels and noncanonical amino acids (ncAAs), while promising, are difficult to apply in live mammalian systems. To overcome these limitations, researchers from the University of Pennsylvania and Children’s Hospital of Philadelphia discovered an innovative intracellular protein editing platform that enables rapid, site-specific, and minimally invasive incorporation of ncAAs and functional handles into endogenous proteins. By integrating split intein-mediated splicing, genetic code expansion, and genome editing, this strategy opens new possibilities for high-resolution, real-time protein studies in live cells.

What is Protein Editing

Protein editing is a posttranslational technique that enables the precise insertion of peptide sequences, including noncanonical amino acids and chemical labels, into proteins that have already been synthesized, either within living cells or in vitro using purified proteins. This approach allows researchers to modify the primary amino acid sequence of endogenous or exogenously expressed proteins at user-defined sites, offering rapid, site-specific, and minimally disruptive manipulation of protein structure and function in their native cellular context.

Inteins, Exteins, and Protein Splicing

Inteins are self-splicing protein segments capable of excising themselves from within a precursor protein and ligating the surrounding sequences, known as exteins, to form a mature, functional protein. This self-removal and ligation process, known as protein splicing, is autocatalytic, meaning it doesn’t require any helper enzymes. The mechanism unfolds in four coordinated steps: peptide bond rearrangement, transesterification, asparaginecyclization to release the intein, and peptide bond formation that fuses the exteins (Fig 1). Depending on the system, this can happen within a single protein chain (cis-splicing) or between two separate chains using split inteins (trans-splicing). Because of their precision and flexibility, inteins have become powerful tools in synthetic biology, especially in protein engineering and modular protein design.

Fig. 1: Intein-mediated protein splicing

Intein-Based Protein Editing Mechanism

Researchers from the University of Pennsylvania and Children’s Hospital of Philadelphia have accomplished protein editing by a combination of the following three main components (Fig. 2):

• Endogenous gene tagging: The target protein is first engineered (often by gene editing techniques like CRISPR-Cas9) to include a short “intein acceptor” sequence at a defined site. The researchers employed two orthogonal split intein pairs: Gp41-1 (splicing half-life ~5 seconds) and AvaX-NpuC (half-life ~1 minute). These inteins were arranged in tandem to enable dual splicing, excising their own domains and inserting a donor peptide into a target protein (Fig. 2).

• Delivery of a donor protein: A recombinant “intein donor” protein, which can be chemically synthesized to include noncanonical amino acids (ncAAs), small-molecule labels, or epitope tags, is introduced into the cell.

• Split intein–mediated protein splicing: A donor is inserted into a target protein at a precise location using a protein trans-splicing reaction, which is a posttranslational reaction where two separate polypeptide chains, each containing a fragment of a split intein, reassemble to catalyze the excision of the intein segments and ligate the flanking external protein sequences (exteins) into a functional mature protein.

Fig. 2: Intracellular protein editing mechanism

This approach allows for the site-specific incorporation of noncanonical residues, chemical labels, or functional domains into proteins in live mammalian cells, including at endogenous expression levels and with temporal control. Importantly, protein editing can be used to install modifications that are not genetically encodable, such as small organic fluorophores, affinity handles (e.g., biotin), or photo-crosslinkers, which are challenging or impossible to introduce by traditional gene editing or genetic code expansion methods.

Editing Model Proteins and Endogenous Targets

As a proof of concept, researchers began by editing fluorescent model proteins in HEK293T cells. They demonstrated that donor payloads, such as an HA epitope tag or the fluorescent protein mRuby3, could be efficiently and specifically spliced onto acceptor proteins like mClover3. The success of this protein editing was confirmed through Western blot analysis, which showed clear evidence of precise and targeted tagging.
The technology was then applied to an endogenous protein, the ER-resident chaperone calnexin. Using CRISPR/Cas9, the researchers inserted the intein acceptor sequence into a cytosolic region of the calnexin gene. When donor proteins carrying an HA tag were delivered into these modified cells, rapid and efficient editing was observed. Mass spectrometry verified the accuracy of the inserted sequence, while interactome analyses confirmed that calnexin maintained its proper cellular localization and biological function following modification.

Generalizability and Functional Preservation

To assess the versatility of this editing platform, researchers tested it on a diverse range of proteins found in various cellular compartments. Cytoskeletal proteins such as β-actin and α-actinin-1 were successfully modified, with the intein-mediated splicing occurring within just 10 minutes of donor delivery. Importantly, these edits did not disrupt the normal localization of the proteins, indicating preserved function.

The system was also applied to the DNA damage response kinase Chk1. A near-traceless editing strategy was used to replace a segment of Chk1 with its native sequence using an intein-containing donor. The edited Chk1 protein retained its phosphorylation response to DNA damage, confirming that its signaling function remained intact. Similarly, editing of the transcription factor c-Myc restored its native sequence from a tagged variant. This enabled time-resolved studies of c-Myc protein turnover without relying on global translation inhibitors. The modified c-Myc exhibited a half-life consistent with previously published data and continued to bind DNA and interact with known regulatory partners.

Incorporation of Noncanonical Amino Acids and Chemical Labels

A key advancement of this platform is its ability to incorporate noncanonical amino acids (ncAAs) and chemical labels into proteins inside living cells. Using genetic code expansion (GCE) in E. coli, donor proteins were engineered to include p-azido-phenylalanine (pAzF), which serves as a bioorthogonal chemical handle for click chemistry. These donor proteins were subsequently labeled in vitro with functional groups such as fluorophores (e.g., TAMRA) or biotin through click chemistry reactions.

Upon delivery into mammalian cells, these modified donors enabled site-specific tagging of endogenous proteins with either fluorescent markers or affinity labels. The success of these modifications was confirmed through fluorescence microscopy, which demonstrated correct subcellular localization, and through pull-down experiments, which allowed antibody-free protein isolation.

A Comparative Overview of Protein Editing and Gene Editing Strategies

Protein editing technologies, such as the intracellular protein editing platform offer distinct advantages over traditional gene editing methods like CRISPR-Cas9 or prime editing. These benefits stem from their posttranslational nature, chemical versatility, and minimal cellular perturbation. A comparison of protein and gene editing techniques are shown in the Table 1.

Table 1: A Comparative Overview of Protein Editing and Gene Editing Strategies

Advantages, Limitations, and Future Directions

One of the key strengths of this protein editing platform is its remarkable temporal resolution, editing can take place within minutes of donor delivery, making it an excellent tool for studying dynamic protein behaviors in real time. The system also minimizes disruption to the target protein; edits leave only a minimal scar, sometimes as small as four amino acids, which helps preserve the protein’s native structure, function, and interactions. Its versatility is another standout feature: it works on both exogenous and endogenous proteins and supports the site-specific installation of a wide range of noncanonical amino acids and chemical labels. The platform is also highly modular and orthogonal, intein pairs are highly specific to one another, and the donor proteins can be labeled with a variety of functional tags in vitro before being introduced into cells. This allows for direct visualization and biochemical tracking of endogenous proteins without the need for antibodies or bulky fluorescent fusion tags, opening up possibilities for cleaner and more accurate analysis.

However, there are some limitations to consider. The method requires an initial genome-editing step to insert the intein acceptor sequence into the target protein’s coding region. While this edit is typically precise, it can potentially affect protein function depending on the site of insertion. Protein delivery is another challenge, although electroporation and lipid nanoparticles have shown good efficiency, improvements in delivery technologies will be critical to expanding the platform’s reach, especially for in vivo applications. Lastly, while the method has shown robust performance across several proteins, these tests have largely focused on loop or disordered regions. Further validation will be needed to determine how broadly the technology can be applied across structurally diverse protein domains.

Reference

Beyer JN, Serebrenik YV, Toy K, Najar MA, Feierman E, Raniszewski NR, Korb E, Shalem O, Burslem GM. Intracellular protein editing enables incorporation of noncanonical residues in endogenous proteins. Science. 2025 May;388(6746):eadr5499. doi: 10.1126/science.adr5499. Epub 2025 May 1. PMID: 40310911.

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