Part:BBa_K2644121
lb-3
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This biological part is specially designed to target a wild type sequence of the second GATA fragment produced by PCR. The principle behind this is that CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems like CRISPR-Cas9. We designed the crRNA which is able to combine with the Cas12a protein and recognize the target DNA. According to this principle, we designed a crRNA that matched with the wild type sequence of the second GATA fragment produced by PCR, which brings Cas12a to its target, specifically cleaves the wild type sequence of the second GATA fragment produced by PCR. Through this method, we are theoretically capable of targeting any single base mutation on ctDNA which are dispersed in people’s blood.
principle of designing crRNA
First, we must put the loop structure which corresponds to the homologous crispr Cas12a as the 5’ side, then put the spacer which length 17-24nt to the 3’ side, the front end of the spacer must have PAM(TTTN) and the mutation side had best at the position of 2-7nt after PAM.
Cleaving experiment
Figure 1.(A) Line 1, GFP plasmid. Line 2, cleavage plasmid with BamH1 enzyme. Line3-8, cleavage according to table B. (B) Experiment design.
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