Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector

Suli Lv; Xuefeng Zhao; Xianyun Ma; Qingli Zou; Neng Li; Yingying Yan; Lidong Sun; Tanjing Song

doi:10.3389/fbioe.2022.960192

Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector

Front Bioeng Biotechnol. 2022 Sep 15:10:960192. doi: 10.3389/fbioe.2022.960192. eCollection 2022.

Authors

Suli Lv¹, Xuefeng Zhao¹, Xianyun Ma¹, Qingli Zou¹, Neng Li¹, Yingying Yan¹, Lidong Sun^{1

2}, Tanjing Song^{1

2}

Affiliations

¹ Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
² Cell Architecture Research Institute, Huazhong University of Science and Technology, Wuhan, Hubei, China.

Abstract

Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.

Keywords: CRISPR; Cas13d; CasRX; SV40; Tet-ON; U6; lentivirus.