# Pro-Codes: A novel vector and cell barcoding technology

> **NIH NIH R33** · ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI · 2020 · $421,587

## Abstract

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 This project will establish a new technology that will enable simultaneous multi-parameter phenotyping of
100s of gene knockouts. This will greatly advance annotation of the genome, and help identify genes important
for numerous facets of cancer biology.
 There are more than 23,000 protein-coding genes in the human genome, as well as 100s of non-coding
RNA genes, including microRNAs. Though there has been progress in assigning some function to many
genes, we still do not know the function of many genes, and we do not know the role of most genes in driving
or affecting disease. Solving this problem requires new technologies that enable high-throughput genetic
screens that incorporate high content phenotyping.
 Over the past few years pooled genetic screens utilizing shRNAs, and now CRISPRs, have emerged as a
powerful and widely used method for identifying genes involved in different aspects of cancer, including cell
growth and drug resistance. Unfortunately, the current technology, which utilizes DNA as a barcode and deep-
sequencing for de-convolution, has major limitations. For one, the read-out is performed on bulk cells, which
means single cells cannot be analyzed with this technology. Another limitation is that DNA barcoding requires
selection of cells based on single phenotypes, predominately cell fitness (do cell's survive or proliferate better).
More informative phenotypes, such as upregulation or downregulation of key proteins, cannot be easily
assessed with current screening technology.
 The objective of this project is to establish a new system of cell barcoding for vector and cell tracking. We
hypothesize that combinations of linear epitopes can be used to generate protein barcodes (Pro-Codes), and
Pro-Codes will enable high content single cell phenotypic analysis of 100s of gene knock-outs simultaneously.
In preliminary studies we established that 10 linear epitopes can be combinatorially assembled to generate 120
different barcodes, which are easily distinguished in cells, at single cell resolution, by FACS and mass
cytometry (CyTOF), with a mere 10 antibodies. We will now: (1) validate and further optimize the Pro-Code
technology for simultaneous single-cell tracking, including generation of sophisticated software for Pro-Code
analysis, (2) establish the Pro-Code technology for CRISPR knockout and multiparameter phenotyping, and
utilize it to identify genes that influence T cell activation, and (3) expand the technology to >1,000 Pro-Codes
and establish their use for double knockout screens.
 The outcome of this project will be a technology that enables high-content annotations of gene functions.
This can help transform cancer research by accelerating the discovery of new drug targets for controlling
cancer growth and immune modulation, as well as identifying mechanisms of cancer biology.

## Key facts

- **NIH application ID:** 9989805
- **Project number:** 5R33CA223947-03
- **Recipient organization:** ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
- **Principal Investigator:** Brian D Brown
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $421,587
- **Award type:** 5
- **Project period:** 2018-09-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9989805

## Citation

> US National Institutes of Health, RePORTER application 9989805, Pro-Codes: A novel vector and cell barcoding technology (5R33CA223947-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9989805. Licensed CC0.

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