Recombinant Marek's disease virus expressing CRISPR/Cas9 targeting Rev and its application
A REV virus, targeted technology, applied in the direction of viruses, applications, antiviral agents, etc.
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Embodiment 1
[0056] Example 1: Design of CRISPR / Cas9 system targeting REV and screening of the best sgRNA
[0057] 1.1 Design of sgRNAs targeting REV
[0058] According to the genome sequence of the REV HLJR0901 strain virus, 25 sgRNAs targeting the U3, R, U5 regions of the REV virus genome and the Pol gene were designed ( figure 1 ). The targeting sequence length of each sgRNA is 20bp, and the downstream is a 3bp NGG (PAM) sequence (Table 1), that is, 5'-N 20 NGG-3' pattern. Using the plasmid pGEM-T-U6 (which contains the sgRNA expression framework, shown in SEQ ID NO.26) as a template, using the primers shown in Table 2, the sgRNAs expression framework was obtained by fusion PCR. The method is as follows: 1) U6F and gLTR1R As a primer, use pGEM-T-U6 as a template to amplify to obtain the gLTR1-1 sequence; 2) use gLTR1F and GEMTR as primers, use pGEM-T-U6 as a template to amplify to obtain the gLTR1-2 sequence; 3) purify The two DNA fragments gLTR1-1 and gLTR1-2 obtained above were us...
Embodiment 2
[0069] Example 2: The effect of CRISPR / Cas9 targeting REV to prevent virus from infecting host cells
[0070] 2.1 Construction of co-expression plasmids targeting REV gRNA and Cas9 gene
[0071] Using pGEM-T-gLTR1 as a template, the gLTR1 expression frame was amplified by PCR, and BamH1 and BglII restriction sites were added at both ends; the gLTR1 expression frame was inserted into the pENTR1 vector through the BglII restriction site to obtain the gLTR1 expression plasmid pENTR1-gLTR1. Using pGEM-T-gLTR6 as a template, the gLTR6 expression frame was amplified by PCR, and BglII and SalI restriction sites were added at both ends; the gLTR6 expression frame was inserted into the pENTR1-gLTR1 vector through the BglII and SalI restriction sites to obtain a gLTR1 co-expression plasmid pENTR1-gLTR1 / 6. PCR amplifies the Cas9 gene (shown in SEQ ID NO.27) and inserts it into the pCAGGS plasmid to construct the Cas9 expression plasmid pCAGGS-Cas9; pCAGGS-Cas9 is digested with SalI and ...
Embodiment 3
[0074] Example 3: Construction and identification of recombinant MDV expressing CRISPR / Cas9 targeting REV
[0075] 3.1 Construction of recombinant mutant cosmid p814-Cas9-gLTR1 / 6 containing CRISPR / Cas9 expression framework
[0076] The CRISPR / Cas9 entry expression plasmid pENTR1-Cas9-gLTR1 / 6 constructed above and the recombinant mutant cosmid p814-5US2KanccdB (for the construction method, please refer to the publication number CN104946678B, the invention name is Marek's disease virus infectious recombinant cloning system and its construction Chinese patent application for method and application) using LR Clonase TM II Enzyme Mix performs LR reaction to replace the Kan-ccdB expression framework in the recombinant mutant cosmid with the CRISPR / Cas9 expression framework in the pENTR1-Cas9-gLTR1 / 6 plasmid, thereby obtaining the insertion of CRISPR / Cas9 into the US2 gene of the MDV genome. Recombinant cosmid p814-Cas9-gLTR1 / 6 ( Figure 5 ).
[0077] 3.2 Rescue of recombinant v...
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