tag:blogger.com,1999:blog-2203096312729800474.post6935472106704183401..comments2022-04-01T10:32:42.048-07:00Comments on A CRISPR blog / bibliography: For #CRISPR HDR, use donor oligos that are complementary to the "gRNA strand". A new paper shows why; see my blog post. Unknownnoreply@blogger.comBlogger7125tag:blogger.com,1999:blog-2203096312729800474.post-86567042183472919222017-01-26T12:32:31.293-08:002017-01-26T12:32:31.293-08:00I actually don't know any off the top of my he...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.Douglas Mortlockhttps://www.blogger.com/profile/11500150356385406841noreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-89056677992688821172017-01-23T02:45:15.981-08:002017-01-23T02:45:15.981-08:00Are you aware of any data on ssODN with mutations ...Are you aware of any data on ssODN with mutations not directly located at the cut site (e.g. 20 bp apart)? <br />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. <br />It would be very interesting to see how that affects homology arms and symmetry.Timnoreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-48678332101970972812016-06-22T13:48:02.332-07:002016-06-22T13:48:02.332-07:00Truly CRISPR-Cas9 was one of the breakthroughs in ...Truly CRISPR-Cas9 was one of the breakthroughs in technology in 2015. It has completely overshadowed ZFNs.Dr. Martin Mulingehttp://biochem.uonbi.ac.ke/noreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-3431109989808495052016-04-18T12:07:48.919-07:002016-04-18T12:07:48.919-07:00I think the mechanism behind that fact is still un...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.Douglas Mortlockhttps://www.blogger.com/profile/11500150356385406841noreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-75150699164483952382016-04-13T08:08:56.030-07:002016-04-13T08:08:56.030-07:00I thought I understood this paper when I first rea...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?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-75667203767297982782016-03-07T07:34:06.682-08:002016-03-07T07:34:06.682-08:00I just wanted to point out that if a quite symmetr...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)!Anonymoushttps://www.blogger.com/profile/10899867207836009708noreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-63387926580202493822016-01-28T20:21:48.504-08:002016-01-28T20:21:48.504-08:00One thing to keep in mind: both the HDR improvemen...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. <br /><br />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?<br /><br />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.<br /><br />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.Anonymousnoreply@blogger.com