Tuesday, July 15, 2014

Current thoughts on targeting reporter cassettes in mice using #CRISPR.

 As an advocate of the PX330 plasmid for implementing CRISPR in mouse zygotes, I am trying to figure out optimal conditions for enabling homology-dependent repair (HDR) with this system.  Genome editing requires HDR, and a donor DNA molecule must be supplied along with the CRISPR/Cas tools such as PX330.  

For introducing "smallish" edits or insertions - like, under 70 bases or so - one can order a single-stranded super-long oligo such as IDT's ultramers.  Larger insertions are going to require double stranded DNA fragments.  Most molecular biologist are well used to purifying these, once they are designed.   Here's a few considerations:

1.  If the CRISPR target is also present in the homology arms of a double-stranded donor, it's probably gonna cut the donor before it gets a chance to donate anything.

2.  What should the optimal ratio of donor/CRISPR/Cas9 be?    I am assuming roughly a 1:1 mass ratio.   My only guidance so far is from the Yang et al 2013 Cell paper.  They were coinjecting donor DNAs with RNAs for guide RNA & Cas9.   Here I will focus on their data for fluorescent reporter fragment insertions, which were injected as circular dsDNA plasmids.   Although this group mainly does cytoplasmic injections, they tested pronuclear injections too.

For pronuclear injections of these reagents. Yang et al  reported 9% and 18% targeting rates, using two different donors and targets.    The concentrations were:  10 ng/µl donor plasmid, 2.5 ng/µl guide RNA, 5 ng/µl Cas9 mRNA.    Thus the total nucleic acid burden of the pronuclear injection material was ~17 ng/µl.   Those with actual experience with mouse embryo injections will note this is rather high;  most DNA transgenes are injected at 1-5 ng/µl, no more.  Why no more?   It can cause toxicity, but even more often and more frustrating, it leads to frequent needle clogging at the injection microscope.   Injected material at these concentrations will need very careful preparation beforehand.  I suggest that a 0.22 micron spin filter be used before each injection over a total 5 ng/µl limit.  Anyway, a ratio of ~1:1 or maybe ~3:2 mass ratio of donor DNA vs. CRISPR reagents seems reasonable.    For PX330 I am starting off by suggesting 4 ng/µl PX330 + 6 ng/µl donor plasmid - although I have no data yet showing whether this is optimal.

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