Hello, I'm studying an article about "In Vivo modelling of ATP1A3 in C. elegans" using CRISPR/Cas9 as a genome editing tool. In the Material and Methods chapter it is reported that they used a sgRNA plasmid as the template to create their own one by amplifying a little portion of U6 promoter and the gRNA scaffold. The linear PCR product was about 100 nucleotides and it is reported that it was circularised using T4 DNA ligase.
I can't understand which kind of plasmid has been generated. Has the PCR amplicon to be inserted in the plasmid? How can you cirularised a 100 nucleotides amplicon?
Sorkaç et al. just created a linearized backbone of the plasmid Addgene #46170 with their primers F and R. For this, these primers are looking in opposite directions/are back to back! Thus, Sorkaç et al. did NOT just amplify a little portion of U6 promoter and the gRNA scaffold.
The process of generating and inserting their own sgRNA fragments into this linearized backbone is described here: Article Rapid and Precise Engineering of the Caenorhabditis elegans ...
Michele Mario Gennari
To modify any plasmid is actually quite simple. You can utilize the multiple cloning site (if present) to use restriction enzymes to cut open the circular plasmid. Designing Primers that contain the same cut overhang as the linearized plasmid, PCR your insert with the cut overhangs included. Take PCR product and linearized plasmid (might need to be gel purified or some reactions are fine without purifying) and set up a ligase reaction and transform the plasmid into your bacteria. Grow your bacteria up, purify plasmid, and test to check if the insert was placed correctly.
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