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.