Part:BBa_K1997010
dCas9
This part codes dCas9 protein.
Usage and Biology
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology. 1
Functional Parameters
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1099
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3378
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through four ways: PCR and functional testing
PCR
Methods
The PCR is performed with Premix EX Taq by Takara.
F-Prime: 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’
R-Prime: 5’- GGACTAGTATTATTGTTTGTCTGCC-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
Functional Test
This part is tested in the composite part BBa_K1997024 and BBa_K1997025.
References
[1] Cong, L.et al.Multiplex Genome Engineering Using CRISPR/Cas Systems,Science 339, 819-823,(2013)
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