Nat Commun. 2023 Jan 26
Shuqian Zhang, Liting Song, Bo Yuan, Cheng Zhang, Jixin Cao, Jinlong Chen, Jiayi Qiu, Yilin Tai, Jingqi Chen,Zilong Qiu,Xing-Ming Zhao, and Tian-Lin Cheng

Products used in the paper Details Operation
AAV vector packaging 5 μl of AAV(1E + 13GC/ml, from PackGene Biotech) were mixed with 5% fast green into the injection syringe. Injection sites at 2/5 of the distance from the lambda suture to each eye were selected for AAV intraventricular injection. Request Quote

Research Field: miniature CRISPR-Cas12f systems

Keywords: CRISPR-Cas9 genome editing, Biotechnology, Molecular engineering

AAV Serotype: AAV9

Dose: 5 μl of AAV(1E + 13GC/ml, from PackGene Biotech) were mixed with 5% fast green into the injection syringe. Injection sites at 2/5 of the distance from the lambda suture to each eye were selected for AAV intraventricular injection. The syringe perpendicular was held to the surface of the skullwith the needle inserted at themarked injection site to a depth of ~3mm. Maximumvolume of 1 μl virus was injected slowly into each ventricle until the dye-filled the ventricle.

Routes of Administration: intraventricular injection.

Targeted organ: brain

Animal or cell line strain: The Postnatal day 0 (P0) C57BL/6 Mus musculus (Vital River Laboratories) was used in this study.

Abstract

Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.

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