# Core D: CRISPRi/a Core

> **NIH NIH U54** · WEILL MEDICAL COLL OF CORNELL UNIV · 2020 · $180,271

## Abstract

PROJECT SUMMARY
The CRISPRi/a core will support research of Projects 1, 2, and 3 by enabling knockdown and overexpression
of endogenous genes in human iPSC-derived neurons. The CRISPRi/a technology, which we co-
developed, enables highly specific, inducible and reversible control of gene expression in mammalian
cells. We use a catalytically inactive version of the bacterial Cas9 protein (dCas9) to recruit transcriptional
repressors (for CRISPRi) or transcriptional activators (for CRISPRa) to endogenous genes, as directed by
single guide RNAs (sgRNAs). We have established the use of this technology in two modes: to investigate the
function of individual genes of interest (reverse genetics), and to conduct genome-wide screens to uncover
genes relevant for a biological process of interest (forward genetics). We will support the research of
Projects 1, 2, and 3 by enabling CRISPRi/a-based forward and reverse genetics in human iPSC-derived
neurons. First, we will generate and validate stable CRISPRi and CRISPRa cell lines from the isogenic human
iPSCs expressing wild-type tau or V337M tau that are used by Projects 1, 2, and 3. Then, we will generate and
validate sgRNAs targeting axon initial segment (AIS) proteins to enable the investigation of their effects on
plasticity and excitability of V337M tau neurons (for Project 1), sgRNAs targeting key autophagy pathways to
address the question if modulation of these pathways can restore neuronal excitability of V337M tau neurons
(for Projects 1, 2, and 3), and sgRNAs targeting proteins selectively interacting with V337M tau that could
underlie the abnormality in neuronal activity and autophagy pathways induced by pathogenic seeding (for
Projects 1 and 3). We will also conduct two genome-wide CRISPRi screens. First, we will aim to identify
cellular pathways controlling tau uptake, which will then be further characterized by Project 2. Second, we will
aim to identify cellular pathways controlling templates tau aggregation. These forward genetics approaches will
complement the hypothesis-driven reverse-genetics approaches and provide an unbiased survey of relevant
cellular pathways. We will work with the Data core to integrate our datasets with those generated by the MS
core, with the goal to identify convergent results from the proteomic and genetic approaches, and to work with
the Human core to validate the relevance of our cell-based findings in human patients.

## Key facts

- **NIH application ID:** 10011935
- **Project number:** 5U54NS100717-05
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Martin Kampmann
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $180,271
- **Award type:** 5
- **Project period:** 2016-09-30 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10011935

## Citation

> US National Institutes of Health, RePORTER application 10011935, Core D: CRISPRi/a Core (5U54NS100717-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10011935. Licensed CC0.

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