CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation.

CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)-based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (similar to 48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (similar to 45% of cholesterol reduction).