Poly(beta-amino ester) nanoparticles enable non-viral delivery of CRISPR/Cas9 plasmids for gene knockout and gene deletion

Abstract The CRISPR/Cas9 system is a powerful gene editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to co-deliver plasmid DNA encoding Cas9 and sgRNA, respectively, to enable gene knockout following 1-cut edits as well as gene deletion following 2-cut edits. We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in iRFP fluorescence while deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing non-viral delivery systems for the CRISPR/Cas9 platform.