Part:BBa_K2148015

gRNA for AGG PAM

This contains an sgRNA which has 20 nucleotide complementary sequences with the Chlamydomonas reinhardtii chloroplast genome upstream of the cut-site, which is at the trnE2-psbH integenic region with psaA promoter. This can be used together with Cas9 to perform site-specific cutting. This part is coded as a Promoter/5UTR/NTAG/CDS/CTAG Phytobrick.

Usage and Biology

This part can be used with a system that manages to express Cas9 protein. It serves as the sgRNA which together with Cas9 can perfrom site-specific cuts on DNA that contains complementary sequences to the target RNA sequence in the sgRNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Alex Reis, Ph.D., Bitesize Bio, Breton Hornblower, Ph.D., Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New England Biolabs, Inc. CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology. NEB expressions Issue I, 2014

John G Doench, Ella Hartenian, Daniel B Graham, Zuzana Tothova, Mudra Hegde, Ian Smith, Meagan Sullender, Benjamin L Ebert, Ramnik J Xavier & David E Root. Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nature Biotechnology 32, 1262–1267 (2014), doi:10.1038/nbt.3026

Matthew H Larson, Luke A Gilbert, Xiaowo Wang, Wendell A Lim, Jonathan S Weissman & Lei S Qi. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols 8, 2180–2196 (2013) doi:10.1038/nprot.2013.132