![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K1218011
Cas9
CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins.
This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.
Usage and Biology
Group: CU-Boulder, 2014
Author: Josephina Hendrix
Summary: CU-Boulder demonstrated that BBa_K1218011 can be targeted to a specific DNA sequence through the modification of its spacer region. The original spacer was replaced with one that targeted a neomycin resistance gene. The modified and unmodified plasmids were transformed into cells containing the targeted gene and the decrease in growth with the target sample demonstrates the ability of the spacer to target a specific sequence.
Documentation:
A 30mer spacer sequence targeting the neomycin phosphotransferase gene was designed and substituted for the original spacer in BBa_K1218011. BW23115 E. coli with the neomycin phosphotransferase inserted into the genome were chemically transformed with the original and modified BBa_K1218011 to compare CRISPR-Cas9 specificity. Transformants were selected for on chloramphenicol.
There was a substantial decrease in growth between the non-targeting (1920 colonies) and the targeting sample (8 colonies) that must be accredited to the differences in spacer sequence. As can be seen in Figure 1B), there is growth in the targeting sample. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.
Group: CU-Boulder, 2014
Author: Josephina Hendrix
Summary: Part BBa_K1218011 was further improved by the addition of the M13 packaging signal (BBa_K1445000) to form the composite part BBa_K1445001. This allows the part to be packaged into M13 phage and delivered to bacteria through phage rather than a transformation method.
Documentation:
The M13 packaging signal was inserted upstream of the CRISPR-Cas9 part and submitted to the registry as part BBa_K1445001. This new part was packaged into phage and introduced via infection to conjugated neomycin resistant bW23115 E. coli. Sample was plated on chloramphenicol to select for cells infected with the phage delivering the phagemid with the Cas9 part and M13 origin of replication.
The growth in figure 2 demonstrates that, with the addition of the M13 origin of replication, BBa_K1218011 can be delivered to cells via recombinant M13 phage.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1642
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3921
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4863
Illegal BsaI.rc site found at 4840
//function/crispr/cas9
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