Efficient, compact, and versatile: Type I‐F2 CRISPR‐Cas system

Abstract

Type I CRISPR-Cas systems are the most prevalent and versatile among CRISPR-Cas systems, widely distributed across prokaryotes 1 . These systems are composed of multisubunit complexes, known as Cascade (CRISPR-associated complex for antiviral defense), which function by binding to CRISPR RNAs (crRNAs) and targeting complementary DNA sequences for degradation. Type I CRISPR-Cas systems, including subtypes I-A to I-G, have been extensively studied for their potential in genome manipulation [2][3][4][5][6][7][8][9][10][11][12] .Although Type I CRISPR-Cas systems offer significant potential for genome editing and transcriptional regulation, they face notable difficulties when used in eukaryotic cells. The main challenge stems from their considerable size and intricate multi-subunit architecture, which complicate their delivery especially when using viral vectors with strict cargo size limits, such as adeno-associated viruses (AAVs). For example, the Cascade complex of the well-studied Escherichia coli Type I-E CRISPR-Cas3 system consists of five subunits with a total gene size exceeding 4.2 kb 7 . Even the more compact Type I-C Cascade from Neisseria lactamica still comprises four subunits, over 3.2 kb in gene size 13,14 (Figure 1A), which is challenging to package and deliver effectively. Moreover, the assembly of these multiple subunits within cells can be inefficient, leading to reduced activity. Furthermore, the lack of a universal protospacer adjacent motif (PAM) across different Type I CRISPR-Cas systems adds another layer of difficulty, as it limits the range of targetable sequences. These limitations highlight the need for more compact and efficient CRISPR systems, particularly for therapeutic applications that require precise and reliable gene editing.The latest study published in Nature Communications presents a milestone in genome editing and transcriptional regulation within human cells, leveraging the smallest Type I system known to date, the I-F2 subtype 15 . Guo et al. focused on overcoming the limitations of traditional Type I CRISPR-Cas systems by developing a minimal version of the I-F2 subtype, derived from Moraxella osloensis CCUG 350. The I-