- Benjamin Gilman 16:48, 11 April 2013 (EDT): I realized that the original page hadn't been addressed properly (It was in CHE391L/S13/Genome_Editing) so I corrected it in the course table of contents and copied all the content from both the main page and the talk section over. Everything should be the same, but the history won't reflect individual changes.
- Kevin Baldridge 17:01, 8 April 2013 (EDT):In the CRISPR/Cas9 section, you refer to crRNAs -- I assume this is cis-repressive, but it might but good to write that out with the abbreviation the first time it's mentioned
- Benjamin Gilman 19:26, 8 April 2013 (EDT): It's a little silly, but crRNA just stands for CRISPR RNA. I made a note of it in the text.
- Catherine I. Mortensen 20:23, 9 April 2013 (EDT):I clicked around on the Targetron page but I couldn't find any specifics on their method of integrating DNA. Do you know how it works? I've never heard of integration without endonucleases, seems interesting.
- Max E. Rubinson 17:44, 10 April 2013 (EDT): With the Targetron system, a precursor RNA containing the group II intron Ll.LtrB and the group II intron-encoded protein LtrA are expressed off of a plasmid transformed into the host. The expressed LtrA protein binds the intron in the expressed precursor RNA and promotes splicing by stabilizing the catalytically active RNA structure (group II introns are technically self-splicing). The resulting ribonucleoprotein (RNP) complex (spliced Ll.LtrB intron bound to LtrA protein) forms base pairing interactions with the DNA target site. Then the intron RNA is inserted through a reaction known as “reverse splicing” into the top strand of the DNA target site while the LtrA protein (which is a multifunctional protein with reverse transcriptase activity) cleaves the bottom strand and then reverse transcribes the inserted intron RNA. The resulting intron cDNA is then fully integrated by the host cell DNA repair machinery. The programmability of introns is based on the fact that you can alter the base pairing interactions the RNP is capable of making in order to insert at a desired locus. More information is available here.
- Benjamin Gilman 19:46, 10 April 2013 (EDT): I changed the header above rAAV and targetrons to be more accurate. As max said, targetrons use the endonuclease activity of the intron-encoded protein LtrA, but unlike ZFNs or TALENs they are targeted by the intron RNA sequence, not by altering a binding domain on the endonuclease. Recombinant AAV uses viral integrase machinery to insert a DNA fragment into sites with homology in the genome. Thus, targeting is controlled by the sequence of a synthetic viral genome, not by altering any proteins.
- Thomas Wall 23:16, 11 April 2013 (EDT): Hey Ben I dunno if this would be workable, but maybe you could do a comparison chart for the major methods (bp's required, range of efficiency in mammals/E. coli). Just think this sort of thing would be good for future reference.
- Benjamin Gilman 14:47, 15 April 2013 (EDT): I intentionally avoided talking about things like range of efficiency and off-targeting rates on the page because they are hugely variable depending on what binding modules are used and what the target site is. Not all ZFN binding domains are created equal, and even TALENs have shown quite a bit of variability depending on target site sequence and cell type. There's a lot of conflicting reports about this (so I'm giving the 'off the top of my head' numbers), but generally the goal for gene targeting in tissue culture cells is >1%, and the actual rates with ZFNs and TALENs are often on the order of 10%. The CRISPER/Cas9 system is really new, so it's not clear how consistent targeting rates are, but they're in the same ballpark. As for off-targeting, ZFNs are generally considered the worst of the three, but that's also up for debate. I included a simple figure that shows the lengths of target sites for each technology and roughly where cleavage occurs. ZFNs are generaly engineered to recognize 12bp per monomer, TALENs can be extended up to 19 modules to bind 19bp per monomer, and CRISPR/Cas9 recognizes 20bp.