Tohoku J. Exp. Med., 2023 May, 260(1)

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1

Luoxi Li,¹ Wenyi Liu,¹ Huacai Zhang,¹ Qingli Cai,¹ Dalin Wen,¹ Juan Du,¹ Jianhui Sun,¹ Li Li,² Chu Gao,¹ Ping Lin,³ Min Wu⁴ and Jianxin Jiang¹

¹Department of Trauma Medical Center, Daping Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Army Medical University, Chongqing, China
²Department of Respiratory Medicine, Daping Hospital, Army Medical University, Chongqing, China
³Biological Science Research Center, Southwest University, Chongqing, China
⁴Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

Key words —— CRISPR-Cas13X; gene knockdown; programmable RNA targeting; RNA editing; RNA methods; type VI CRISPR‐Cas systems

© 2023 Tohoku University Medical Press

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Tohoku J. Exp. Med 2023 May, 260(1), 51-61.

Correspondence: Jianxin Jiang, Department of Trauma Medical Center, Daping Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Army Medical University, Chongqing 400042, China.

e-mail: hellojjx@tmmu.edu.cn