Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR-Cas type V-I systems utilize Cas12i nucleases and crRNA for DNA targeting.
• Existing systems often require a tracrRNA, limiting their versatility.
• The Cas12i effector's compact nature and self-processing capabilities offer potential for improved delivery and multiplexing.

Purpose of the Study:

• To engineer an enhanced Type V-I CRISPR system (Cas12i) for improved genome editing.
• To develop a tracrRNA-less Cas12i system, designated ABR-001, with high efficiency and specificity.
• To evaluate the ABR-001 system's performance in various human cell types and in vivo models for gene therapy applications.

Main Methods:

• Unbiased mutational scanning was employed to enhance the editing activity of an initial Cas12i2 variant.
• The engineered ABR-001 system was tested for genome editing in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells.
• Adeno-associated virus (AAV) vector delivery was used to assess ABR-001 editing efficiency in HEK293T cells.

Main Results:

• The engineered ABR-001 variant demonstrated broad genome editing capabilities across tested human cell types.
• ABR-001 exhibited robust editing efficiency and high specificity in cellular and ex vivo models.
• High-level genome editing was achieved with ABR-001 when delivered via AAV vector in HEK293T cells.

Conclusions:

• The engineered Cas12i system, ABR-001, represents a versatile and high-performance platform for gene editing.
• ABR-001's tracrRNA-less design and compact size facilitate diverse delivery strategies for gene therapy.
• This study establishes ABR-001 as a promising tool for both ex vivo and in vivo gene therapy applications.