# Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9

> **NIH NIH P20** · BROWN UNIVERSITY · 2021 · $377,377

## Abstract

Project Summary
Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular
biology has linked numerous pathologies to dysregulation of these processes. Although modification of
the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot
be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR-
Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations,
however, to fully realize these applications, novel strategies to overcome its off-target effects and poor
temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and
facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif
(PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9
nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer
little mechanistic information, as the target DNA and catalytic nucleases are never observed in an
activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring
subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an
allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision
medicine by establishing novel paradigms to control and enhance the spatial and temporal function of
Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease
and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric
network that controls Cas9 function. To investigate the reach of this allosteric network and the role of
molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and
computational study to map the long-range allosteric pathway of Cas9. We will now (1) characterize
allosteric mutants of HNH that are known to alter Cas9 specificty, (2) establish the biophysical roles of
the neighboring REC2 and REC3 domains in propagating allosteric signals to/from HNH, and (3)
characterize the conformational ensemble governing the full-length Cas9 protein. This multidisciplinary
approach of NMR spin relaxation experiments, molecular dynamics simulations, and network theory, will
probe multi-timescale protein motions in Cas9, revealing specific amino acids responsible for transmitting
structural or dynamic information. These studies will use both full-length Cas9 and novel engineered
constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic
findings of this work will be correlated to function with new in vivo assays to provide a detailed
understanding of the Cas9 allosteric mechanism.

## Key facts

- **NIH application ID:** 10271625
- **Project number:** 2P20GM109035-06
- **Recipient organization:** BROWN UNIVERSITY
- **Principal Investigator:** GEORGE LISI
- **Activity code:** P20 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $377,377
- **Award type:** 2
- **Project period:** 2016-06-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10271625

## Citation

> US National Institutes of Health, RePORTER application 10271625, Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9 (2P20GM109035-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10271625. Licensed CC0.

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