Part:BBa_K2556061
sgRNA-cm
Single-guide RNA (sgRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It contains the 20-bp complementary region (N20) with the requisite NGG PAM matching genomic loci of interest and the sequence of tracrRNA. It will guide the nuclease Cas protein 9(Cas9) to the target. The N20 of sgRNA-cm is complementary to the 20 bp fragment of the chloramphenicol gene. When cas9 protein is present, sgRNA-cm will guide cas9 to cleave the chloramphenicol resistance gene.
Functional verification
The strain we used is E.coli MG1655 wild type. First, we need to prepare E.coli MG1655 wild type competent cells and transform pSu plasmid(there is chloramphenicol resistance gene) into it. Then, we need to culture the transformants and prepare them into competent cells, and then transform pCas plasmid(express cas9 gene). Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid(contain BBa_K2556061), respectively. Finally, the transformed plates were placed in 30℃ devices overnight.
sgRNA-cm can guide Cas9 to the cm resistance gene and cause double-strand DNA break in the pSu plasmid. After the transformed plates placed in 30℃ devices overnight, E.coli MG1655 wild type still has pCas plasmid and pTargetF-cm plasmid. So it can grow in LB+kan+spec. However the pSu plasmid can’t work. So it died in LB+cm.
Experimental Results
After final transforming and the transformed plates placed in 30℃ devices overnight. We selected the strain and culture it both in LB+kan+spec and LB+cm. And the strain grew in LB+kan+spec, while it died in LB+cm（Fig.1）.
Figure 1. The result of culture
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]
References
[1]Jiang W, Bikard D, Cox D, et al. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 2013, 31(3):233-239.
[2]Citorik R J, Mimee M, Lu T K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases[J]. Nature Biotechnology, 2014, 32(11):1141-1145.
[3]Didovyk A, Borek B, Hasty J, et al. Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9[J]. Acs Synthetic Biology, 2016, 5(1):81-88.
[4]Doench J G, Fusi N, Sullender M, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9[J]. Nature Biotechnology, 2016, 34(2):184-191.
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