Tuesday, July 22, 2014

Hard numbers re: eGFP and CRE knock-ins with #CRISPR in rat zygotes!

This one has important implications for CRISPR-mediated insertion of fragments in the several-kilobase size range, such and GFP, CRE, etc. , into rats - and likely, mice - via pronuclear injection.  

Generation of eGFP and Cre knockin rats by CRISPR/Cas9.   FEBS Journal.  Accepted manuscript online: 17 JUL 2014.   

  • Yuanwu Ma1
  • Jing Ma1
  • Xu Zhang1
  • Wei Chen1
  • Lei Yu1
  • Yingdong Lu1
  • Lin Bai1,
  • Bin Shen2
  • Xingxu Huang2,* and
  • Lianfeng Zhang1

  • A brief summary from a quick dig I made into this paper:  They did 3 different CRISPR targeted kncok-ins, that is, using HDR to insert cassettes into genes of interest.  The cassettes were GFP and CRE into 1 and 2 different genes respectively.  This was all done by pronuclear injection into rat zygotes, of RNAs for Cas9 and guide RNA plus circular, double-stranded DNA plasmids containing the cassettes of interest.    So this is technically, essentially the same process one would use for mice.   Their numbers were quite impressive:   between 23% to 54% of live pups carried the targeted insertion.

    A key ratio to scrutinize is the ratio of targeted insertions to the total number of insertions and other mutations, e.g. indels.   The reason is that the total number reflects the number of pups in which CRISPR clearly had activity.   Therefore the ratio reflects the proportion of the time that the HDR process occurred successfully as a subset of all embryos that had some sort of CRISPR-mediated cleavage event.  Since almost all of their live pups had evidence of the HDR insertions or indels anyway, the ratio of targeted/all events is still about 25-50%.   

    How big were the homology arms used?   Between ~1.5 to 2.1 kb in all cases (3 constructs x 2 arms each = 6 arms total).    Concentrations used were:   25 ng/µl Cas9 mRNA, 10 ng/µl, and 4 ng/µl of the donor plasmid in circular form.   

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