Immunology and Infection
Published: October 30th, 2016
Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.
The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.
Vaccinia virus (VV) is an enveloped DNA virus belonging to the poxvirus family and has played a crucial role in one of the greatest achievements in medicine of the eradication of smallpox. In the post-eradication era of smallpox, VV has been developed as a vector for delivering genes for vaccines against HIV and other infectious diseases1-7 by inserting genes from various pathogens into VV vectors. VV has also been extensively used as a vector for cancer immunotherapy8-14 especially for the development of tumor-targeted replicating oncolytic vaccinia virus. In order to create an efficient vaccine vector with improved selectivity for cancer cells, modifications within the viral genome are required, including gene deletions or introduction of therapeutic genes.
With the development of DNA technology and a better understanding of molecular biology and virology, insertion of foreign DNA into VV was originally achieved using homologous recombination (HR) in 1980s 15. This method is still widely used for constructing VV vectors. Introduction of the genetic modification is achieved by using a shuttle vector for HR, which recombines with the VV genome in pre-infected cells. However, this method has proven to be highly inefficient (less than 1% homologous recombination efficiency16) and often results in random insertion of the selection marker into non-targeted regions and/or loss of the marker upon virus expansion.
The efficiency of DNA homologous recombination for inserting exogenous DNA at genomic loci can be dramatically enhanced in the presence of double-strand breaks (DSBs) 17. Therefore, the technology that can induce DSBs at target loci holds great potential for genome engineering of VV.
The recently developed CRISPR-Cas9 system shows promise for triggering DSBs in any VV gene region. CRISPR-Cas9 is an RNA-guided nuclease involved in adaptive immunity against invading phages and other foreign genetic materials18-20. There are three CRISPR-Cas systems in a range of microbial species21. The type II CRISPR-Cas system is widely used for editing the genome of eukaryotic cells and large viruses. It consists of the RNA-guided Cas9 endonuclease (from Streptococcus pyogenes), a single guide RNA (sgRNA) and the trans-activating crRNA (tracrRNA) 22-24. The Cas9/sgRNA complex recognizes the complementary 20-nucleotide genomic sequence preceding a 5'-NGG-3' Protospacer adjacent motif (PAM) sequence in mammalian cells22, 23. It has been successfully used for effective generation of genetically modified cells, viruses and animal models22-32.
The CRISPR-Cas9 system has proven to be an efficient tool for genome targeting in combination with homologous recombination in the cytoplasm of VV infected CV-1 cells to generate mutant vaccinia viruses33, 34. In order to extend the potential application of this method, we present detailed information about this system's methodology. The described protocol can be used to create a mutant VV with a particular gene deletion and/or arm the mutant virus with a therapeutic transgene.
1. Preparation of Target RNA and Cas9 Constructs and Repair Donor Vector
2. Seeding Cells (Day 1)
3. Transfection of Cas9 Plasmid and gRNA Plasmid (Day 2)
4. Infection of CV-1 Cells and Shuttle Donor Vector Transfection for HR (Day 3)
5. Harvest the Recombinant Virus (Day 4)
6. Purification of the Modified VV (First Round)
7. Purification of the Modified VV (Second Round)
8. Further Rounds of Plaque Purification
9. Expand the Purified Plaque(s)
10. Verification of the Modification of Mutant VV Using PCR
For construction of a new VV vector, the key starting point is to design and construct a donor shuttle vector that can target a particular region of the genome. In this study, the VV N1L gene was used as an example target. 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 recombination33. One day (24 h) post-transfection, RFP was expressed in the CV-1 cells (Figure 2). After infection of CV-1 cells with the whole lysate from the homologous recombination (step 5), RFP-positive and negative plaques were both observed in the CV-1 cells (Figure 3). Following the purification protocol described, a pure plaque could be obtained after 3-5 rounds of purification. As shown in Figure 4, all plaques after infection with cell lysate derived from a pure plaque with the mutant vaccinia virus presented an RFP signal. To verify whether the pure mutant VV had the correct gene modification, PCR was used to confirm the absence of the targeted N1L gene, as shown in Figure 5. PCR of the modified virus showed a positive signal for RFP and an absent signal for N1L (Figure 5 lane A-G), while the PCR results of N1L for the control virus (Ctr) with an intact N1L region is positive. The control DNA fragments of A52R were positive for all recombinant viruses and the Ctr virus. The HR efficiency in RFP-positive plaques is 100% (6/6) in this experiment (it was up to 94% in the previous work34). The method described herein has improved the recombination efficiency by more than 100 fold compared to conventional protocols33, 34.
Figure 1: The cassette of the donor shuttle vector for the homologous recombination, and the targeted region in the VV genome. The purification marker gene RFP is driven by the H5 promoter. The repair donor vector targeting the N1L region results in the RFP gene being incorporated into the N1L region after homologous recombination. 'X' indicates the homologous sequence in the repair donor vector that will replace the same sequence on the vaccinia virus genome. Please click here to view a larger version of this figure.
Figure 2: Imaging of the CV-1 cells one day after homologous recombination. One day after homologous recombination, the cells infected with VV and transfected by repair donor vector are RFP-positive. A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3: An RFP-positive plaque in the first round purification of mutant virus. In the first-round purification of the mutant virus, the RFP-positive plaque (circled with red line) with the target region deletion are surrounded by plaques formed by wild type virus (circled with yellow line). A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: The pure mutant VV. After 3-5 rounds of purification, pure plaques were obtained as all the plaques were RFP-positive. A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 5: Verification of the pure mutant VV. DNA was extracted from VV infected CV-1 cells. 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 work33, 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. Please click here to view a larger version of this figure.
There are two main goals regarding modification of VV for therapeutic purposes. One is to delete a particular gene, such as the thymidine kinase (TK) gene to attenuate virus for anti-tumor therapeutic use. The other is to arm VV with a desired therapeutic gene (such as GM-CSF) or a marker gene (such as luciferase gene). Achieving either of these involves the deletion of a target region/gene. A direct DNA ligation method35 and an in vitro packaging method36 have been used previously to generate mutant vaccinia viruses with TK gene modifications. However, these methods have limited application regarding mutation of other regions in the genome due to a lack of unique restriction enzyme sites across the genome. The current approach to creating mutant VV involves transfection of a shuttle (donor) vector with a selection marker (RFP or GFP) into CV-1 cells two hours after infection with VV to induce a homologous recombination incorporating the selection marker into the target region. Selection of marker-positive plaques is followed by their purification 24 h post-transfection. The plaque purification process can take up to 10 rounds, last 3-4 weeks and often leads to undesired recombinant viruses with selection markers inserted in off-target regions. DNA double-strand breaks can effectively induce homologous recombination in mammalian cells 37, and by harnessing this mechanism, the efficiency of generating mutant VV can be vastly improved. The gRNA-guided Cas9 system has been successfully employed in genome editing and enhances the efficiency of homologous recombination in eukaryotic organisms22, 25. Recently, in host cells the genomes of adenovirus and type I herpes simplex virus were edited by the gRNA-guided Cas9 system 24. It has been recently demonstrated that CRISPR Cas9 system is a powerful tool for efficiently editing the VV genome and constructing a VV vector for expressing a particular gene.
Both N1L gRNA-guided Cas9 and the traditional homologous recombination (HR) method resulted in successful HR events at the same target site of N1L. Interestingly, however gRNA-guided Cas9 induced HR at much higher levels of efficiency than the traditional HR method. 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 system33. 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. 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 efficiency33, 34.
The limitation for the CRISPR/Cas9 in editing VV is the low rate of RFP-positive plaques in the first round of recombination. To obtain a sufficient number of RFP-positive plaques containing recombinant virus, usually at least ten 6-well plates are needed, which makes first-round screening tedious. Hence, there is still a need to optimize the protocol to overcome this limitation.
The small size of RFP-positive plaques is another potential problem, which is due to a high volume of lysate used to infect a 6-well plate. To overcome this, a smaller volume of lysate should be used to infect a 6-well plate to obtain larger size RFP-positive plaques. Using a smaller volume of lysate will also enable for better separation of RFP-positive plaques from the RFP-negative ones. Consequently fewer rounds of plaque purification are required to obtain pure RFP-positive plaques.
Off-target effects are always an unwanted issue in gene editing. CRISPR-Cas9 has shown off-target effects. However, with careful design of gRNA, off-target effects can be avoided when editing the VV genomes33, 34. This is the particular benefit of using the gRNA-guided Cas9 system for editing the genome of VV. Given the promises of VV, using the CRISPR/Cas9 system will speed up the development of mutant VVs for biomedical research and in translational medicine. In addition, such a system would also expedite discoveries in cell biology, such as dissection of the signaling pathways used by VV for its actin-based motility.
Authors have no competing financial interests.
This work was supported by The MRC DPFS grant (MR/M015696/1) and Ministry of Sciences and Technology of China (2013DFG32080).
|gRNA cloning vector
|Cas9 cloning vector
|repair donor vector cloning
|One Shot TOP10 Chemically Competent E .Coli
|QIAprep Spin Miniprep Kit
|Dulbecco’s Eagle’s Medium (DMEM)
|cell culture medium
|fetal bovine serum (FBS)
|cell culture serum
|Thermo Scientific Nalgene Cryogenic vial; 2.0 mL
|Olympus BX51 Fluorescence
|find RFP-positive plque
|DNeasy Blood & Tissue Kit
|Extensor Long PCR ReddyMix Master Mix
|10cm culture dish
|hold cell suspension
|N1L forward 5’-TATCTAGCAATGGACCGT-3’
|N1L reverse 5’-CCGAAGGTAGTAGCATGGA-3'
|A52R forward 5’-ATAGGATTGTGTGCATGC-3’
|A52R reverse 5’-TTGCGGTATATGTATGAGGTG-3’
|RFP forward 5’- GCTACCGGACTCAGATCCA-3’
|RFP reverse 5’-CGCCTTAAGATACATTGATGAG-3’
|resolve PCR product
|UV gel doc
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