Efficiently Editing the Vaccinia Virus Genome by Using the CRISPR-Cas9 System

Abstract

dVaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. Since the eradication of smallpox, vaccinia virus (VACV) has been developed as a vector for vaccines against infectious diseases and immunotherapies for cancer (1-4), including oncolytic virotherapies (5-8). The renewed interest in VACV has driven a number of vaccine and therapeutic candidates to clinical trials, showing especially encouraging results for cancer treatment (5, 7). To improve VACV as a vector for vaccine or cancer therapy, a flexible system is required to delete viral genes or arm the VACV with therapeutic genes. Such a system would also expedite discoveries in cell biology, such as dissection of the signaling pathways used by VACV for its actin-based motility (9). Several strategies have been developed to construct VACV vectors (10-12); the current method for modification of VACV is based on homologous recombination in mammalian cells, but only 1% to 5% of the recombinant plaques contain the inserted DNA (13).The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system is a natural microbial immune mechanism against invading viruses and other genetic elements (14-16). The CRISPR-Cas9 system, consisting of the RNA-guided Cas9 endonuclease (from Streptococcus pyogenes), a single guide RNA (sgRNA), and the trans-activating CRISPR RNA (tracrRNA), has been adapted for genome editing in eukaryotic cells (17,18). The system has been used successfully for efficient generation of genetically modified cells and animal models (17,(19)(20)(21). Recently, the genomes of adenovirus and type I herpes simplex virus were edited using the gRNA-guided Cas9 system (22). We hypothesized that the CRISPR-Cas9 system could specifically generate doublestrand breaks (DSBs) in the target DNA sites of VACV, increasing the efficiency of editing VACV genomes and constructing new VACV vectors expressing therapeutic genes.Given that VACV replicates in the cytoplasm of infected cells, the Cas9 gene without the nuclear localization signal (NLS) was cloned into the pST1374 vector (Fig. 1A). The expression of Cas9 was confirmed (Fig. 1A). Given the important role of the N1L gene in virulence and in regulating the host immune response to VACV (23-25), we chose this as an example to validate the application of CRISPR-Cas9 for editing VACV. Two individual sgRNAs targeting the VACV N1L (LO24) gene were cloned into this guide RNA (gRNA) cloning vector (Fig. 1B) and designated gRNA N1 and N2. To prove that the CRISPR-Cas9 system could improve the efficiency of constructing a VACV vector expressing a therapeuti...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

“…The HR efficiency in RFP-positive plaques is 100% (6/6) in this experiment (it was up to 94% in the previous work 34 ). The method described herein has improved the recombination efficiency by more than 100 fold compared to conventional protocols 33,34 . The deletion of the target region N1L was verified by PCR using N1L primers, the insertion of RFP into N1L region was confirmed by PCR using RFP primers.…”

“…The deletion of the target region N1L was verified by PCR using N1L primers, the insertion of RFP into N1L region was confirmed by PCR using RFP primers. Lanes A-F correspond to six purified plaques, lane G is a control plaque with N1L deletion verified in previous work 33 , lane Ctr (control) is the wild type vaccinia virus (with N1L region intact). A52R gene was amplified as the control gene for all the samples.…”

“…The cassette of the donor shuttle vector for the recombination and the targeted region in the VV are shown in Figure 1. In order to enhance the efficiency of the homologous recombination, plasmids expressing Cas9 and a N1L-specific gRNA were co-transfected into the CV-1 cells 48 h prior to performing the homologous recombination 33 . One day (24 h) post-transfection, RFP was expressed in the CV-1 cells (Figure 2).…”

“…The 24 h interval allows Cas9 and gRNA to be expressed at a reasonable level. Furthermore, the gRNA sequence targeting a particular gene may need to be optimized as we found that the different gRNA sequences targeting N1L gene varied in their efficiency 33,34 .…”

“…Thus, the use of gRNA-guided Cas9 eliminates the need to purify as many plaques to obtain mutant VVs. In this work, we significantly improved the efficiency and time-frame for generation of mutant VV using the gRNA-guided Cas9 system 33 . Of note, one critical step for obtaining a high efficiency of homologous recombination is to transfect CV-1 cells with the plasmids expressing Cas9 and the gRNA for 24 h before performing the conventional homologous recombination.…”