Single-gRNA adenovirus CRISPR vector
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
U6 Promoter: This drives high level expression of the downstream gRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
gRNA: Guide RNA compatible with Cas9 derived from Streptococcus pyogenes.
Terminator: Terminates transcription of the gRNA.
CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
Dual-gRNA adenovirus CRISPR vector
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
U6 Promoter: This drives high level expression of the downstream gRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
gRNA #1: The first guide RNA compatible with Cas9 derived from Streptococcus pyogenes.
gRNA #2: The second guide RNA compatible with Cas9 derived from Streptococcus pyogenes.
Terminator: Terminates transcription of the gRNA.
CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.
Cas9 protein: The open reading frame of the Cas9 nuclease is placed here.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.