Establishing CRISPR-Cas9 in the sexually dimorphic moss, Ceratodon purpureus.

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.

Unraveling the role of the enigmatic MatK maturase in chloroplast group IIA intron excision.

Maturases are prokaryotic enzymes that aid self-excision of introns in precursor RNAs and have evolutionary ties to the nuclear spliceosome. Both the mitochondria and chloroplast, due to their prokaryotic origin, encode a single intron maturase, MatR for the mitochondria and MatK for the chloroplast. MatK is proposed to aid excision of seven different chloroplast group IIA introns that reside within precursor RNAs for essential elements of chloroplast function. We have developed an in vitro activity assay to test chloroplast group IIA intron excision. Using this assay, we demonstrate self-excision of the group IIA intron of the second intron of rps12 and the group IIA intron of rpl2. We further show that the addition of heterologously expressed MatK protein increases efficiency of group IIA intron self-splicing for the second intron of rps12 but not the group IIA intron of rpl2. These data, to our knowledge, provide the first direct evidence of MatK's maturase activity.