Fig. 3

Toolkit for cloning CRISPR adenoviruses. a Multiple candidate sgRNAs targeting cancer genes of interest are cloned into plasmids co-expressing Cas9 and a puromycin resistance gene for functional validation in cell culture. b Selected validated sgRNA expression cassettes including U6 promoter and sgRNA scaffold are PCR amplified using primer pairs adding a BbsI recognition site and a 4-bp motif specifying the position in the final vector construct. c Optional cloning of PCR amplicons for sequence verification by Sanger sequencing. d Release of complementary overhangs by BbsI. e Golden Gate assembly of multiple BbsI-digested sgRNA-cassettes with BsaI-digested shuttle vectors containing expression cassettes for Cre + Cas9 or Cre only. f Gateway recombination cloning of modified sgRNA-containing shuttle vectors into the adenoviral vector backbone (pAd/PL-Dest destination vector). g Release of linear adenoviral DNA by PacI digest. h Transfection of Ad293 cells for production and amplification of infectious AV particles