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Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants. [ABSTRACT FROM AUTHOR]

An original Agrobacterium tumefaciens-mediated transformation procedure, based on the actions of both wild type and disarmed bacterial strains, was developed. The aux2 gene of A. rhizogenes was introduced into a rapid-cycling genotype of cabbage ( Brassica oleracea L.). The aux2 gene product converts naphthalene acetamide into the auxin naphthalene acetic acid. Expression of this gene in the transgenic progeny grown in vitro led to an altered root phenotype. On a medium supplemented with napthalene acetamide (NAM), two of the three analysed progenies were characterized by the formation of callus instead of roots, whereas on a NAM-free medium all the plantlets from these progenies presented a normal phenotype. Expression of the aux2 gene was also assessed under horticultural conditions by sowing seeds in sand and watering them with a nutritive solution supplemented with NAM. Under these conditions, NAM inhibited the formation of a root system in transgenic plantlets and induced the death of the transgenic plantlets three to four weeks after germination. Thus, aux2 acts as a lethal conditional marker which could be used in negative selection of cabbage. Potential utilization of the aux2 gene to screen spontaneous androgenetic plants in order to transfer cytoplasmic male sterility in a single generation is discussed. [ABSTRACT FROM AUTHOR]