BBa_K1818000 T7-BbsI-gRNA
This is a cassette for the expression of a custom guide RNA for use in CRISPR-Cas9 genome editing. It may be utilized in vivo or amplified via PCR for in vitro transcription. This parts consists of a T7 promoter, dual oppositely-oriented BbsI cut sites, the guide RNA scaffold, and a terminator. Since BbsI cuts outside its recognition sequence, overhangs are created at the end of the T7 promoter and the start of the gRNA scaffold. A custom 20 nucleotide targeting sequence can be installed here if it shares the same overhangs. To do this, we recommend annealing two oligonucleotides, phosphorylating them, and ligating that insert into this part. The T7 promoter starts transcription at it second to last base pair. This means all transcripts will start with GG. The genomic target should therefore also start with GG (not to be confused with the distal NGG protospacer adjacent motif, which is also essential). A protocol for oligo design and insertion can be found here: