Vaccinia virus (VACV) continues to be used in immunotherapy for prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method for 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 gRNAs targeting all VACV genes will be valuable for the study of VACV gene functions.
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