I need help designing homology arms for CRISPR - (Jun/02/2014 )
I am relatively new to cloning. I want to design a neomycin cassette (dsODN) with specific 40-60bp overhangs (one overhang on either stranded). I want to transfect my cassette in with the CRISPR nickase that cleaves single stranded DNA about 40nt apart (exacly like Figure 5a in Cell 154, 1380–1389, September 12, 2013, except with neomycin)
I have no idea how I would go about doing an overhang PCR reaction for homologous arms. Could some help me or provide a protocol?
If you only need 40-60bp on each side of the cassette, you could easily design oligos containing the homology arm sequences followed by the Kan cassette-specific forward and reverse sequences. Do pcr with a high fidelity polymerase (i use NEB's Phusion), column purify the pcr reaction, quantify and go :-)
(You will probably want to run a bit of the pcr on a geek to make sure you don't have nonspecific bands. If you do, gel purify the correct band before using it)
Oh and make sure that the CRISPR site spans the split between the two arms. If the site is present intact in one arm or the other, it will get cut as well and your efficiency will suck.