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

• Published in: Nature communications Vol. 13; no. 1; p. 2833
• Main Authors: McGaw, Colin, Garrity, Anthony J., Munoz, Gabrielle Z., Haswell, Jeffrey R., Sengupta, Sejuti, Keston-Smith, Elise, Hunnewell, Pratyusha, Ornstein, Alexa, Bose, Mishti, Wessells, Quinton, Jakimo, Noah, Yan, Paul, Zhang, Huaibin, Alfonse, Lauren E., Ziblat, Roy, Carte, Jason M., Lu, Wei-Cheng, Cerchione, Derek, Hilbert, Brendan, Sothiselvam, Shanmugapriya, Yan, Winston X., Cheng, David R., Scott, David A., DiTommaso, Tia, Chong, Shaorong
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 20.05.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 38/47; 42/109; 42/41; 49/23; 49/31; 631/337/4041/3196; 631/61/201; 692/4017; CD34 antigen; Cell lines; CRISPR; CRISPR-Cas Systems - genetics; Editing; Endonucleases - genetics; Endonucleases - metabolism; Gene Editing - methods; Gene therapy; Genome editing; Genomes; gRNA; HEK293 Cells; Hematopoietic stem cells; Humanities and Social Sciences; Humans; Lymphocytes; Lymphocytes T; Mammalian cells; multidisciplinary; Multiplexing; Nuclease; Progenitor cells; RNA - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Scanning; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-30465-7

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy. Cas12i is a genome editing platform with compact size that fits in AAV vector with short 43-mer gRNA, absence of tracrRNA, ability to process pre-crRNA, and high specificity. Here the authors present an unbiased mutational scanning approach to engineer Cas12i, which shows low activity in mammalian cells, and identify single substitutions that significantly improve indel activity.