# (PQ4) Novel tools for in vivo study of genetic interactions in cancer progression

> **NIH NIH R01** · YALE UNIVERSITY · 2022 · $122,682

## Abstract

PROJECT SUMMARY:
The evolution of human cancer is a complex process driven by multiple molecular and cellular events. We
endeavored to harness single-effector RNA-guided endonucleases (RGNs) for genome editing, parallel
screening and in vivo modeling of human cancer. Recently, we generated a platform to systematically
interrogate several hundred loci directly in vivo. To overcome current limitations in multigene editing and
achieve more accurate control of simultaneity and sequentiality of multi-allelic tumor modeling, we utilized
Cpf1, an RGN that can edit its target simply with crRNAs independent of tracrRNA thus allowing simultaneous
editing of multiple genes with a single crRNA array. We developed a preliminary Cpf1-based crRNA array
screening (CCAS) system in mammalian cells, and applied it in mouse models of progression and metastasis.
In our first aim, we will perform validation and optimization of CCAS for in vivo double-knockout phenotyping of
cancer co-drivers. We will establish its technical rigor, efficiency and specificity for simultaneous editing, as
well as developing a set of computational pipelines for accurate calling of statistically significant gene pairs. We
will apply this approach to study the genetic interactions of tumor suppressors found in lung cancer patients at
Yale Cancer Center and Hospital, and identify potential co-drivers of metastasis to vital organs. In the second
aim, we will carry out validation and optimization of a Cpf1-Flip system for sequential mutagenesis of cancer
targets. We will demonstrate its broader applicability by testing clinically relevant gene sets identified from
public studies of the genomics of metastasis as well as a large multi-sample metastasis dataset gathered on
Yale cancer patients. We will then apply this methodology as an unbiased depletion screen to identify targets
that are essential for survival in specific oncogenic backgrounds. We will develop novel versatile transgenic
mouse strains and companion viral vectors for direct modeling of multigenic tumorigenesis in mice. We will
combine these tools to enable high-throughput genetic interaction screening in healthy cells directly in the
native organ to identify causative mutation pairs that drive tumorigenesis. We anticipate that developing and
establishing these tools will transform multigenic tumor modeling and pre-clinical studies of human cancer,
directly addressing NCI Provocative Question 4. These powerful toolkits will enable scientists to target any
gene pairs or combinations simultaneously or sequentially, assessing the phenotypic outcome of their in vivo
interactions in tumor progression, metastasis, synthetic lethality, drug sensitivity or other processes in cancer
evolution. This supplement’s goal is to (1) extend and strengthen the existing research described in Aim 3; and
to (2) promote diversity in health-related research by involving an under-represented minority trainee in the
project. This will mostly involve Aim 3...

## Key facts

- **NIH application ID:** 10599597
- **Project number:** 3R01CA231112-05S1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Sidi Chen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $122,682
- **Award type:** 3
- **Project period:** 2018-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10599597

## Citation

> US National Institutes of Health, RePORTER application 10599597, (PQ4) Novel tools for in vivo study of genetic interactions in cancer progression (3R01CA231112-05S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10599597. Licensed CC0.

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