CRISPR-Cas systems, particularly type II Cas9 and type V Cpfl (Cas12a), are valuable tools for therapeutic gene editing. To expand this toolbox, researchers identified functionally diverse type V systems (V-G, V-H, and V-I). Cas12i, a subtype V-I system, is evolutionarily distinct but functionally similar to Cas12a, processing pre-crRNA without needing tracrRNA. Its compact size and short crRNA make it ideal for multiplexed editing and viral delivery. However, Cas12i1 and Cas12i2 had not previously shown therapeutic utility in mammalian cells. This study aimed to engineer Cas12i2 to overcome this limitation and develop a highly efficient and specific genome editing platform for therapeutic applications, addressing the need for improved CRISPR systems suitable for both ex vivo and in vivo gene therapies.

The literature review focuses on the existing CRISPR-Cas systems, specifically highlighting the advantages and limitations of Type II (Cas9) and Type V (Cas12a) systems. It emphasizes the potential of Type V-I systems (Cas12i) due to their compact size and tracrRNA-independent nature. However, the review also notes the lack of success in utilizing Cas12i for therapeutic genome editing in mammalian cells, setting the stage for the current study's focus on engineering Cas12i2 to enhance its activity and address this gap.

The researchers employed a high-throughput mutational scanning approach to engineer Cas12i2. Initially, they utilized an in vitro assay coupling cell-free protein synthesis with a fluorescent reporter assay to screen a large number of arginine and glycine substitutions within the C-terminal RuvC domain. This high-throughput screen allowed for rapid identification of variants with enhanced activity. Promising variants from the in vitro screen were then tested in HEK293T cells for indel activity using targeted deep sequencing. The most effective substitutions were combined to create ABR-001. The specificity of ABR-001 was assessed using tagmentation-based tag integration site sequencing (TTISS) and an in silico prediction approach. Ex vivo editing capabilities were evaluated using human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs) using ribonucleoprotein (RNP) delivery via electroporation. In vivo studies involved transplanting ABR-001-edited HSPCs into immunodeficient mice. Finally, the potential for AAV vector delivery was investigated by constructing an AAV2 vector expressing ABR-001 and measuring indel rates in HEK293T cells.

Wild-type Cas12i2 showed low editing efficiency in mammalian cells. The in vitro screen identified three key substitutions (D581R, I926R, and V1030G) that, when combined in ABR-001, significantly enhanced indel activity in HEK293T cells, approaching the efficiency of SpCas9. ABR-001 demonstrated high specificity, with fewer off-target sites identified compared to SpCas9 using both TTISS and in silico prediction methods. ABR-001 RNPs effectively edited therapeutically relevant genes in primary human T cells and CD34+ HSPCs without compromising cell viability. In CD34+ HSPCs, ABR-001 induced fetal hemoglobin (HbF) expression at levels comparable to SpCas9, despite lower indel rates and GATAA motif disruption. In vivo studies showed persistent editing and similar or higher engraftment capacity of ABR-001-edited cells compared to SpCas9-edited cells. Finally, ABR-001 delivered via AAV vector achieved highly efficient genome editing in HEK293T cells, reaching indel rates comparable to SaCas9.

The successful engineering of Cas12i2 into ABR-001 demonstrates that improving the interaction of the CRISPR effector with mammalian genomic DNA can significantly enhance its editing efficiency. The high specificity of ABR-001 suggests it is a safer alternative to existing systems. The ability to achieve high editing rates in primary cells, particularly HSPCs, and the successful AAV delivery showcase its therapeutic potential for both ex vivo and in vivo applications. The findings suggest that the broad deletion profile of ABR-001, particularly beneficial for disrupting non-coding elements, makes it a uniquely advantageous tool for gene editing. Further optimization using structural information might lead to even higher efficiency and specificity.

ABR-001, an engineered Cas12i2 variant, offers a versatile, efficient, and specific platform for therapeutic genome editing. Its compact size, tracrRNA independence, pre-crRNA processing capability, and high performance, especially with AAV delivery, make it a promising candidate for both ex vivo and in vivo gene therapies. Future research could focus on further optimization of ABR-001, exploring its applications in a wider range of therapeutic targets and disease models.

While the study demonstrates ABR-001's effectiveness, the in vivo experiments were conducted in immunodeficient mice, potentially overestimating the therapeutic outcome in immunocompetent individuals. Further studies in relevant animal models are necessary to validate these findings. The comprehensive assessment of off-target effects might be limited by the methods used, warranting further investigation to completely characterize its off-target profile.