A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Hye Jin Shin,Heon Seok Kim,Chonsaeng Kim,Soojin Kim,Bum-Tae Kim,Seong-Jun Kim,Yeon-Soo Kim,Keun Bon Ku

doi:10.3390/v13020237

Abstract

Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics.

Highlights

The type II clustered regularly interspaced short palindromic (CRISPR) system has been used for gene-specific modification and functional genetic screens [1,2,3,4,5]
Coxsackievirus B3 (CVB3) titers in the pancreas were similar between sg-acyl-CoA binding domain containing 3 (ACBD3) and sg-Cont injected mice (Figure 1E). These results suggest that the delivery of single-guide RNAs (sgRNAs) using ssAAV8 does not efficiently induce the mutations to investigate the effect of ACBD3 on the pancreatic infection of CVB3
These results suggest that the delivery of sgRNAs using ssAAV8 d4 oofes13 not efficiently induce the mutations to investigate the effect of ACBD3 on the pancreatic infection of CVB3

Summary

Introduction

The type II clustered regularly interspaced short palindromic (CRISPR) system has been used for gene-specific modification and functional genetic screens [1,2,3,4,5]. Cas can be combined with single-guide RNAs (sgRNAs) to generate DNA double-strand breaks, which activate the DNA repair system for insertions or deletions (indels) of DNA sequences This genetic perturbation introduced the loss-of-function mutations and revealed the functional role of specific genes. The AAV vector systems contain a single-stranded (ss) genome that is converted to a double-stranded (ds) form prior to transgenic expression This conversion is a slow process responsible for delayed transduction and inefficiency [13,14]. To facilitate more versatile applications in in vivo mouse experiments, a Cas knock-in mouse was developed [16] These Cas9-expressing mice showed no toxicities and tolerated the overexpression of the Cas protein [17]. This mouse line, along with AAV, expressing only sgRNAs, can be applied to achieve loss-of-function mutations in murine tissues efficiently without the limitation of a packaging capacity

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