Thursday, January 28, 2016

For #CRISPR HDR, use donor oligos that are complementary to the "gRNA strand". A new paper shows why; see my blog post.

(ERRATUM:  I made on correction to this post on March 11 2016.  In the original version of the post I stated that the paper implied that the donor oligo should have "additional length of homology on the PAM-distal side as compared to the PAM-proximal side".  That was a mistake - it turns out the opposite was true.   The authors found that additional length of homology on the PAM-proximal side was favorable.  I got confused because the PAM in figure 3 is on the "bottom" strand, not the top, so the PAM-proximal side is to the left of the cut site in their oligo schematics.  Figure 3 has an "upside down" Cas9 icon in keeping with this fact.  Thank you Scot for letting me know about the error!).

After a long break from blogging... here's a nice nugget of insight about CRISPR-mediated homologous recombination.    Back in late 2014 I blogged about 
Optimal design of ssODNs (donor oligos) for #CRISPR - length and strandedness data? . In that post I pointed out a curious observation that HDR oligos work "better" when they are designed from the strand that is complementary to the protospacer/gRNA sequence.   This was somewhat counterintuitive to me, as one might think that in a complementary HDR oligo would tend to anneal to the gRNA, reducing its availability or kinetics somewhat and generally interfering with Cas9's job.   But empirically, this was not the case; complementary oligos work better.

Now, Richardson et al seem to have found an explanation. (Richardson et al, Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nature Biotechnology (2016, Published online 20 January 2016)). Turns out that following double-stranded DNA cleavage by Cas9, the first component of the DNA molecule that it releases is the 3' end of the DNA strand that corresponds to the protospacer/gRNA.  Quote: Hence, although Cas9 globally dissociates from duplex DNA in a symmetric fashion (Fig. 1c), it appears that the enzyme locally releases the PAM-distal nontarget strand after cleavage but before dissociation."  And here is a nice diagram from the supplemental material of the paper that shows how this strand "breathes" after cleavage.  I thank the senior author, Jacob Corn, for graciously allowing me to reproduce the image here:

(OK - before moving forward, let's get clarity on the terms here; when we're comparing the two strands of the DNA containing the CRISPR target, the "target" strand is the DNA strand that directly will anneal to the gRNA.  Thus, the "target strand" is complementary to the gRNA.  The non-target DNA strand encodes the protospacer and the "NGG" PAM sequence.   Got it? )   

Read the above quote again.  The first bit of DNA that is released by Cas9 is the single-stranded 3' end of the non-target strand.  This immediately suggests 2 things:

1.  Cas9 releases the non-target strand before the target strand.  Thus the non-target strand is available sooner than the target strand to potentially engage with a complementary donor molecule and jump-start homologous recombination.      

2.  The 3' end of the non-target strand, which is "PAM-distal", is released first.  This also suggests that the design considerations for homology might be different for the PAM-distal and PAM-proximal sides of the cleavage location.

For this second point, the practical consideration is that commercially available ssDNA oligos are usually limited to 200 bases or less (depending on the vendor's capabilities; 120 base oligos can work well too).  So, we are limited in the length of homology we can actually apply to each side of the cleavage site.   Instead of centering the oligo (e.g. for a 120 base HDR oligo = ~60 bases of homology on each side) it may be better to skew the oligo design to have more homology on one side of the cut site versus the other.  That is what Richardson et al found - at least for one target they investigated in detail  (See Fig. 3c, d, e.)

Here's another thought. The authors note that Cas9 actually stays on the DNA for quite a while after it cleaves both strands - about 5 hours.    This might have something to do with why DNA repair takes significantly longer on Cas9-cleaved breaks than on breaks induced with radiation - Cas9 may just sitting there, sterically hindering the DNA repair proteins from accessing the free DNA ends at the break.  Perhaps, CRISPR mutagenesis efficiencies could be further enhanced by increasing Cas9's intrinsic off-rate?  On this note, another recent paper by Kleinstiver et al (Keith Joung lab) suggests that directed mutations can destabilize Cas9's non-specific interaction with DNA.   The authors note that this reduces off-target cleavage significantly while preserving on-target cleavage .   While they did not see dramatic increases in on-target efficiency, perhaps in some experimental contexts there might be?    Hmm.    


  1. One thing to keep in mind: both the HDR improvement and the Joung lab modification (which is basically the same as the earlier Zhang lab directed evolution paper: excusable if both labs were not in the same place or had similar businesss) were done in cell lines that are particularly odd with respect to repair efficiency. It will be interesting to see if either of these strategies pans out in more primary-like or stem-like cells or in embryo injections.

    As for the rate of repair, the literature shows that oddly enough two-guide-based deletions in human cells result in highly precise joining of the genomic junctions kinda of independently of which way the guides are facing. Maybe the non-target strand is released but still occluded?

    If Cas9 is sterically hindering repair, there were easier ways to remove it. There's already two direct papers and one indirect paper about destabilizing Cas9 to get rid of it. All three use some variant of the FKBP12 domain.

    Another possible interpretation is Cas9 and the DNA repair machinery just keep going through cycles of cutting followed by precise repair until the precise NHEJ machinery just gives up.

  2. I just wanted to point out that if a quite symmetric oligo is used, this effect is strongly or completely diminished (figure 2 c At versus An) or inverted if the oligo is designed in the opposite way (Dt - Dn)!

  3. I thought I understood this paper when I first read it, but looking back in light of your correction I'm now confused. Why does maximizing PAM-proximal homology improve efficiency? Isn't it the distal strand that's released first by Cas9 (as in the figure you show), freeing it up for annealing with the ssOligo?

    1. I think the mechanism behind that fact is still unclear. One possibility is that because the PAM-distal "top" strand is released first it may actually be "easier" for donor strand invasion and pairing to occur on that side; therefore, increased homology on the PAM-distal side , relative to the length of homology on the PAM-proximal side, appears beneficial. I have no idea if this idea holds water though.

  4. Truly CRISPR-Cas9 was one of the breakthroughs in technology in 2015. It has completely overshadowed ZFNs.

  5. Are you aware of any data on ssODN with mutations not directly located at the cut site (e.g. 20 bp apart)?
    For some loci it is quite difficult - if not impossible - to get Cas9 cut exactly at the site where you want to introduce the change.
    It would be very interesting to see how that affects homology arms and symmetry.

    1. I actually don't know any off the top of my head. For the dozen or so designs involving ssODNs that I have done, I always have been able to find an overlapping target, but I think that it just luck. If I see examples I will try to comment about them on this post.