# Nano-Polymers as Cas9 Inhibitors

## TL;DR

Researchers found that certain nano-polymers can inhibit the Cas9 enzyme, which could improve the precision of CRISPR gene editing.

## Contribution

A new class of biodegradable nano-polymer Cas9 inhibitors is introduced for potential use in CRISPR delivery.

## Key findings

- Polymers like PMLA, PGA, and PLD conjugated with LLL inhibit Cas9 in a dose-dependent manner.
- PMLA-LLL binds directly to Cas9, reducing its affinity for sgRNA.
- Molecular docking and microscopy suggest a possible mechanism for Cas9 inhibition.

## Abstract

Despite wide applications of CRISPR/Cas9 technology, effective approaches for CRISPR delivery with functional control are limited. In an attempt to develop a nanoscale CRSIPR/Cas9 delivery platform, we discovered that several biocompatible polymers, including polymalic acid (PMLA), polyglutamic acid (PGA), and polyaspartic acid (PLD), when conjugated with a trileucine (LLL) moiety, can effectively inhibit Cas9 nuclease function. The Cas9 inhibition by those polymers is dose-dependent, with varying efficiency to achieve 100% inhibition. Further biophysical studies revealed that PMLA-LLL directly binds the Cas9 protein, resulting in a substantial decrease in Cas9/sgRNA binding affinity. Transmission electron microscopy and molecular docking were performed to provide a possible binding mechanism for PMLA-LLL to interact with Cas9. This work identified a new class of Cas9 inhibitor in nano-polymer form. These biodegradable polymers may serve as novel Cas9 delivery vehicles with a potential to enhance the precision of Cas9-mediated gene editing.