NEW YORK (GenomeWeb) – To overcome the challenges of limited phenotyping and bulk-cell resolution that can constrain gene function research performed using DNA barcodes, researchers at the Icahn School of Medicine at Mount Sinai have developed a barcoding system operating at the protein level. And by pairing each protein barcode with a different CRISPR guide RNA, they were able to achieve simultaneous high-dimensional protein-level phenotyping of hundreds of genes with single-cell resolution.
In a study published online today in Cell, the researchers reported that they synthesized modules encoding triplet combinations of linear epitopes to generate more than 100 unique protein barcodes which they dubbed Pro-Codes. They then introduced Pro-Code-expressing vectors into cells and analyzed them by CyTOF mass-cytometry.
"Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts," the authors wrote. "Pro-Code/CRISPR screens found two interferon-stimulated genes, the immuno-proteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells, and identified Socs1 as a negative regulator of Pd-l1."
The researchers began by selecting 10 linear epitopes that have antibodies that can be detected, including epitopes commonly used as protein tags, such as HA and FLAG. They synthesized the DNA sequence encoding each epitope and assembled them in every possible combination of three, for a total of 120 different combinations, and fused each epitope combination to dNGFR, a truncated receptor without an intracellular domain, which is commonly used as a reporter protein. These were dubbed Pro-Codes. Using an expanded set of 14 epitopes, the team then generated 364 three-epitope Pro-Code vectors.
Through various experiments, the investigators found that the Pro-Codes are not differentially rejected and can be used in vivo. They can also be used in cell-tracking studies, they noted.
Importantly, one of the advantages of the Pro-Codes is that they can be used to enable protein-level phenotyping in genetic screens. To test this possibility, the team generated 96 CRISPR gRNAs targeting 54 different genes and paired each gRNA with a different Pro-Code. The researchers then analyzed 500,000 cells by CyTOF. All 96 Pro-Code populations were resolved and clustered, which enabled them to examine expression of the surface proteins on each of the 96 Pro-Code/CRISPR populations with single-cell resolution. They found that Pro-Codes can mark cells encoding a specific CRISPR gRNA and can enable protein-level phenotyping in pooled CRISPR screens.
In one specific experiment, the researchers set out to determine if they could use Pro-Codes to identify genes conferring cancer cell sensitivity or resistance to T cell immunity. They generated a library of 56 CRISPR gRNAs targeting 14 different genes and paired each CRISPR with a unique Pro-Code, forming a pool of 56 Pro-Code/CRISPR vectors. They selected the 14 genes to contain known regulators of immunity, such as B2m, and several genes with no known role, such as Cldn4. As a model of breast cancer, they utilized the 4T1 mammary carcinoma cell line.
They found that knock-out of two IFNg-inducible genes, Psmb8 and Rtp4, resulted in resistance to antigen-specific T-cell killing. Psmb8 is a component of the immune-proteasome and its expression has been found to positively correlate with the abundance of tumor-infiltrating lymphocytes in breast cancer, the researchers reported. Rtp4 is a chaperone protein involved in folding G protein-coupled receptors. Its only defined targets are opioid receptors, and despite being an IFN-stimulated gene, almost nothing is known about the role of Rtp4 in immunity.
While the Pro-Codes that currently exist are far fewer than those that have been created with DNA, the authors concluded, it is an order of magnitude greater than what currently exists with protein reporters. "Moreover, 1,000s of new Pro-Codes can be created by applying the principle we used here and introducing additional epitopes and epitope positions," they added. "Although generating genome-wide Pro-Code/CRISPR libraries cannot be done at the relative ease of DNA barcoded libraries, the Pro-Code will primarily be for more focused screens; concentrating on specific pathways or gene classes and targeting 100 to 500 genes."
They also noted that it could be possible to create Pro-Code/CRISPR libraries with non-overlapping epitopes and use them together in complex screens to identify cooperating or redundant genes as a greater number of epitopes are validated.
Importantly, they said, Pro-Codes provide the ability to perform high-dimensional phenotyping of multiple proteins in pooled screens, which was not feasible with DNA barcodes. "As DNA barcode de-convolution is generally performed on bulk cells, this means cells with complete, partial, or no [knock-out] are lumped together in the analysis," the authors wrote.
"With Pro-Codes, every cell expressing a CRISPR is analyzed individually. Even when the targeted gene itself is not analyzed, the phenotypic differences can be seen between individual cells receiving the same CRISPR," they added. "This provides a powerful advance to pooled screening."