Part:BBa_K2813001
SpCas9 with gRNA
This part is a Cas9 nuclease enzyme derived from Streptococcus pyogenes (Sp) that is able to cleave DNA at highly specific sites based on a guide RNA sequence. In nature, it serves as a small part of the larger CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system which acts an immune system for the bacteria against bacteriophages. Specifically, Cas9 serves to seek out and cut the sequence that corresponds to the guide sequence given to it in the form of the guide RNA (gRNA). Additionally, it has a T7 RNA polymerase promoter (BBa_J64997), a rrnB T1 terminator (BBa_B0010), and a T7 Terminator (BBa_B0012), to control the translational frame and protein expression.
The LacI repressor protein is used to inducibly control the expression of the Cas9 protein in the plasmid. The LacI repressor protein will bind to the LacI operator sequence, which is upstream of the Cas9 promoter allowing expression to occur in the presence of allolactose or IPTG.
The gRNA target Sequence corresponds to the DNA sequence to be cut. In our case, we included a guide sequence that corresponds to a reporter plasmid containing GFP but this gene can be modified with a double digest reaction. The gRNA Sp scaffold will connect to the gRNA and serves to stabilize the RNA within the Sp Cas9 enzyme. The gRNA Sp scaffold sequence is specific to the enzyme and will remain constant regardless of target sequence.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1953
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1506
Illegal NheI site found at 1529
Illegal NheI site found at 3119 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1586
Illegal BamHI site found at 5398 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1953
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1953
Illegal NgoMIV site found at 1851 - 1000COMPATIBLE WITH RFC[1000]
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