Easy cloning of CRISPR/Cas9 Binary Vectors
The binary vectors contain Cas9 (nuclease or nickase) and the gRNA. Cas9 is codon optimized for high expression in monocots and dicots. To avoid problems with the nuclease activity of Cas9 in bacteria, the first intron from Nicotiana tabacum rbcs is inserted.
Cas9 nuclease vector for single and multiple target sequences
RNA is driven by the U6 promoter from Arabidopsis thaliana (for dicots) or Oryza sativa (for monocots). Insertion of the target RNA in the Cas9 nuclease vector can be easily done as primer into the BsmBI restriction site of the binary vector. Cloning of multiple target sequences can also be done into the BsmBI site. For further informations see the nickase construct.
Cas9 nickase vector
Insertion of the target RNA in the Cas9 nickase vector can be easily done also into the BsmBI restriction site of the binary vector as DNA fragment containing both target RNAs at the ends. The inserted DNA fragment containing the U6 promoter , the RNA scaffold and both target RNAs at the ends, can be easily amplified from a plasmid provided with the starter kit or synthesized.