Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.
RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Edward M
EM
Kennedy
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Anand V R
AVR
Kornepati
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Adam L
AL
Mefferd
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Joy B
JB
Marshall
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Kevin
K
Tsai
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States.
Hal P
HP
Bogerd
Department of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, United States. Electronic address: bryan.cullen@duke.edu.
Bryan R
BR
Cullen
D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use*; D064113:CRISPR-Cas Systems; D000229:Dependovirus / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D011401:Promoter Regions, Genetic; D013211:Staphylococcus aureus / Q000235:genetics; D014777:Virus Diseases / Q000235:genetics / Q000628:therapy*
D000998:Antiviral Agents
AAV multiplex genome editing; CRISPR/Cas9; Quantitative sgRNA screening method; Staphylococcus aureus Cas9; Viral therapeutic; tRNA promoters