CasMINI: The next generation of genome engineering

CRISPR-Cas systems have revolutionized genome engineering, offering opportunities for the development of gene therapies against a variety of human diseases. However, their large sizes often restrict delivery into cells, impeding clinical utility. For instance, Cas9 and Cas12a—nucleases capable of specific genome editing—are typically 1,000-1,500 amino acids, which doesn’t leave much room for guide RNAs (gRNA) and regulatory elements when considering the packaging capacity of adeno-associated viral vectors (<4.7 kb).      

So, how can we get CRISPR-Cas systems into cells to do their job? Make them smaller. This is easier said than done. While smaller naturally occurring Cas effectors exist (e.g., Cas12f and Cas12j), they are not necessarily functional in mammalian cells. Xu et al., explore smaller Cas proteins to develop more compact—and functional—CRISPR-Cas systems and, ultimately, overcome cell delivery challenges.

By optimizing the single guide RNA (sgRNA) design and performing multiple rounds of iterative protein engineering and screening Xu et al., generated Cas12f variants to obtain:

• Nuclease-deactivated CasMINI (dCasMINI) that, when fused with a transcriptional activator, could activate reporter and endogenous gene expression;
• Nuclease-deactivated CasMINI with base editing capabilities; and
• Nuclease-active CasMINI with efficient and distinct genome editing capabilities.

Xu et. al developed a miniature CRISPR system using Cas12f for genome engineering that functions robustly in mammalian cells. The engineered CasMINI is compact and less than half the size of Cas9 and Cas12a.

Gene Activation

The researchers generated a dCasMINI with significant improvement in gene activation over the wild-type dCas12f system and comparable activation ability with the dCas12a system (see the table below for specific mutations and their impact). Through whole-transcriptome RNA sequencing, the researchers found that dCasMINI is specific in mammalian cells (comparable with dCas12a).

All of these mutations reside in the DNA binding pocket.

With an optimized sgRNA, dCasMINI efficiently activated genes endogenous to HEK293T cells including HBG, IL1RN, ASCL1, IFN????, CD2, CXCR4, and HBB. What’s more, dCasMINI outperformed dCas12f for endogenous gene activation and is comparable with dCas12a. For example, IFNg activation with dCasMINI reached a 300-fold improvement over dCas12f on the mRNA level and 768-fold on the protein level when co-delivering two sgRNAs.

Base Editing

By fusing dCasMINI to the deoxyadenosine deaminase domain heterodimer (TadA-TadA-8e), the researchers observed detectable A-T to G-C base conversion at several genomic sites by using high-throughput sequencing. Base editing efficiency was dependent on the target site and was most efficient 3-4 bp downstream of the PAM.

Gene Editing

Using nuclease-active versions of dCasMINI variants (CasMINI), the researchers observed

robust gene editing. Specifically, adding an additional mutation (E151A) to CasMINI-V3 resulted in more efficient indel formation at four genomic sites in HBB and IFN???? compared to wild-type Cas12f and CasMINI-V2. Additionally, nuclease-active CasMINI resulted in distinct editing patterns from Cas9.

Xu, Xiaoshu, et al. "Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing." Molecular Cell (2021). https://doi.org/10.1016/j.molcel.2021.08.008