CRISPR/Cas9

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

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.

amp pcrbinary nick 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.

New Genes for TALENs

For cloning of TALENs we have developed a binary vector which harbors both DNAs on one plasmid. Cloning  of the  TALE repeat domain can be done either in the SacI-BbvCI or into the AscI-HindIII restriction sites. The present vector contains the Ubi-maize promoter for transformation of monocots. But the TALENs cassette can be simply transfered with SfiI restriction to any other binary containing these restriction sites. Both  backbones of the TALENs construct were codon optimized for high expression in monocots and dicots. They are identical in amino acid sequence. But to  avoid problems due to repeated sequences, both backbones are not identical in the sequence of the nucleic acid. Both 35s promoters can be easily exchanged. We recommend small promoters, since cloning can be difficult, when the complete construct reaches 20 kb.

P7iU

Synthetic Genes

Genes used in plant molecular biology sometimes have low expression in plants due to the fact that they have been originally isolated from bacteria which use a different codon usage.
The beta­glucuronidase (GUS) gene from E. coli was codon optimized for better expression in monocots and dicots. To prevent expression from bacteria the StLS1 intron was introduced. The synthetic GUS gene is regulated by the Ubi promoter from maize and introduced into maize embryos by particle gun bombardment. For comparison the GUS gene from Jefferson et al. (EMBO J. 87 Dec) was used, which is 100% identical in amino acid sequence and in the sourrounding of the genes.

     

GUS gene
(Jefferson et al. 87)
Optimized Gus gene
by adapted codon usage
Vector