Hello,
I wish to knock-in a gene to a certain locus of a mammalian cell line using an sgRNA, Cas9, and HDR template. I will first transduce cells with the sgRNA and Cas9 in a lentiviral vector. Following selection with antibiotic, I will transfect in my sslDNA HDR template. Will this order of steps work? Or does the sgRNA/Cas9 need to be expressed simultaneously with the HDR template so that homologous recombination can occur immediately after the Cas9 cut? If cells constitutively express sgRNA/Cas9 then is the target sequence constantly cut until the sgRNA cannot recognize its template?
Thank you.
Hello david;
I don't have pratical experience, but as you, I'm going to start a knock-in experiment.
Studying the literature and speaking with experts I understand that you have to introduce in your cells, at the same moment the three components:
1)the guide,
2)the cas9
3)the donor (the new sequence).
To avoid that your integrated donor sequence will became a Cas9 target, you could introduce silent mutation changing some 3th bases.
If you do not want to change your donor sequence, you should avoid system with stable cas9 expression. You could use or pcdna-vector or purified cas9 protein.
regards
Hello,
you should have all three components at the same time in the cells. If you express Cas9/sgRNA first, a lot of cells will accumulate mutations in the vicinity of Cas9 cleavage site because of NHEJ. I would recommend having all three experimental parts on the same expression vector if possible or transfected at the same time. I would also synchronize cells in S or early G2 phase to facilitate HDR over NHEJ. I like to insert specific endonuclease site in my knock-ins so I can check for successful knock-in by doing a simple PCR product digestion.
Hi,
you are correct. If your cells constantly express gRNA and Cas9 it will make indels at the target. The process of HDR happens only when you provide a donor template and it should be available during the process of double-stranded break (DSB) repair. When the cellular machinery finds the donor template after the DSB, it inserts or creates a complementary sequence and corrects the DSB with the sequences from the donor template.
So, the HDR experiment can only work when you transduce the gRNA+Cas9 and donor constructs simultaneously. Then subject the cells to antibiotic selection for a few days. From the antibiotic-resistant cells, either do a single-cell sorting or serial dilution to get single-cell clones. Take a small fraction of cells from individual clones and do junction PCR. If you want the data from the pool, do NGS or ddPCR.
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