Paper: Recombination products suggest the frequent occurrence of aberrant gene replacement in the moss Physcomitrella patens by Wendeler et al.
This paper examines in detail what happen around the PpCOL2 locus of Physcomitrella patens during gene replacement experiments. They find that complex re-arrangements were very frequent. In particular, a number of transformed lines where PCR analysis across the predicted genome / replacement construct junctions was positive had other copies of the target locus in the genome. They find that this is RAD51-dependent (there are two non-redundant RAD51 genes in Physco, -a and -b).
The main take-away for me in this paper was that PCR analysis of junctions is insufficient to screen for gene replacement lines in Physco. For instance, in their southern blotting experiments, the authors find that “gene replacement with correct recombination junction fragments and deletion of the original sequence was obtained in only two [sic] out of 9 targeted lines” .. where these “9 targeted lines” were all positive for both junction PCRs. Yikes. The southern blot is in figure 3B.
This matches with our own past experiences of gene replacement in Physocmitrella. It is not trivial to actually get rid of targeted sequence. Perhaps with a-miRNAs and also perhaps CRISPR-Cas9, gene replacement in this organism might be deprecated.
– Mike Axtell