Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

TLDR

Compact and versatile CRISPR‑Cas systems will enable genome engineering applications through high‑efficiency delivery in a wide variety of contexts. The study aims to develop an efficient miniature Cas system (CasMINI) for genome regulation and editing. CasMINI was engineered from the type V‑F Cas12f (Cas14) system via guide RNA and protein modifications, producing a system less than half the size of Cas9 or Cas12a. CasMINI achieves high levels of gene activation (up to thousands‑fold), matches Cas12a activity, is highly specific, supports robust base editing and gene editing, and is expected to be broadly useful for cell engineering and gene therapy ex vivo and in vivo.

Abstract

Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.