tag:blogger.com,1999:blog-2203096312729800474.post6064356523269181594..comments2022-04-01T10:32:42.048-07:00Comments on A CRISPR blog / bibliography: Move over, Cas9: Cpf1 may be your new #CRISPR competition.Unknownnoreply@blogger.comBlogger3125tag:blogger.com,1999:blog-2203096312729800474.post-73999509630428224012015-10-05T14:41:02.464-07:002015-10-05T14:41:02.464-07:00I feel like the efficiency for HDR or even targete...I feel like the efficiency for HDR or even targeted insertion will be terrible with this system. If you think about it, "sticky ends" was already tried with the Cas9 nickases and the resulting efficiency was pretty bad (I also heard it worked worse than regular Cas9-mediated HDR in embryos, but that was never published). <br /><br />To me, the most likely order of events is: (1) Cpf1 cuts and generates "sticky ends" (2) the "sticky ends" are almost immediately chewed back on by Artemis or some other endogenous nuclease (3) the now blunt ends are ligated back together by DNA ligase 4. <br /><br />The fact that the biggest theoretical advantage of this system doesn't appear to have been attempted in the paper (increased HDR or targeted insertion) is a huge concern.<br /><br />It's also possible Cpf1 will just sit there for nearly an eternity (on the DNA-binding protein timescale) just like Cas9. Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-13209645141181065282015-09-30T11:01:03.570-07:002015-09-30T11:01:03.570-07:00Yes - i agree that it may and the authors refer to...Yes - i agree that it may and the authors refer to this in the discussion. Although in this case, the edit will definitely have to include the silent mutations to disrupt the target and hopefully the efficiency will still hold up. Douglas Mortlockhttps://www.blogger.com/profile/11500150356385406841noreply@blogger.comtag:blogger.com,1999:blog-2203096312729800474.post-72730363794878847832015-09-30T10:54:59.248-07:002015-09-30T10:54:59.248-07:00Hey Doug,
Do you think the fact that the cut site...Hey Doug,<br /><br />Do you think the fact that the cut site is distal to the PAM will make this system more efficient for HDR driven editing? I wonder if, because small indels won't immediately disrupt target cleavage, then it may be that the Cpf1 will remain active in the system until HDR mediated repair occurs, and the target site is removed by silent mutations this way...Anonymoushttps://www.blogger.com/profile/10053963862068477116noreply@blogger.com