Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons

Yuxi Chen; Shengyao Zhi; Puping Liang; Qi Zheng; Mengni Liu; Qi Zhao; Jian Ren; Jun Cui; Junjiu Huang; Yizhi Liu; Zhou Songyang

doi:10.1016/j.omtm.2020.05.011

Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons

Mol Ther Methods Clin Dev. 2020 May 22:18:33-43. doi: 10.1016/j.omtm.2020.05.011. eCollection 2020 Sep 11.

Authors

Yuxi Chen^{1

2}, Shengyao Zhi², Puping Liang², Qi Zheng², Mengni Liu², Qi Zhao², Jian Ren², Jun Cui², Junjiu Huang^{1

2

3}, Yizhi Liu¹, Zhou Songyang^{1

2}

Affiliations

¹ State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
² MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
³ Key Laboratory of Reproductive Medicine of Guangdong Province, The First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

Abstract

Herpes simplex keratitis (HSK) is the most common cause of corneal blindness in developed nations, caused by primary or recurrent herpes simplex virus 1 (HSV-1) infection of the cornea. Latent infection of HSV-1, especially in the trigeminal ganglion (TG), causes recurrence of HSV-1 infection. As antiviral treatment is not effective on latent HSV-1, to test the possibility of inhibiting HSV-1 by SpCas9 (Streptococcus pyogenes Cas9) or SaCas9 (Staphylococcus aureus Cas9), ICP0 and ICP4, two important genes required for HSV-1 replication and reactivation, were chosen as targets. In Vero cells, SpCas9 and SaCas9 targeting ICP0 or ICP4 can effectively inhibit the proliferation of HSV-1 without affecting cell viability. No significant guide RNA (gRNA)-dependent off-targets were observed in the human genome by digenome sequencing and deep sequencing verification. Adeno-associated virus 1 (AAV1)-mediated delivery of SaCas9 inhibits HSV-1 replication by targeting ICP4 in mouse primary TG neuronal cells. SpCas9 and SaCas9 are able to inhibit HSV-1 infection in Vero cells and mouse TG neuronal cultures with high efficiency and good biosafety. AAV1-mediated delivery of SaCas9 shows great potential in treating HSK and inhibiting HSV-1 in TG neurons. Further investigations may be needed to test the inhibition of latent infections, which may result in the development of novel methods for treating viral diseases.

Keywords: AAV; HSK; HSV-1; SaCas9; trigeminal ganglion neuron.