Pervasive generation of oppositely oriented spacers during CRISPR adaptation. Nucleic acids research. 2014; 42:5907–5916. doi:10.1093/nar/gku226. [PubMed: 24728991

During the process of prokaryotic CRISPR adapta-tion, a copy of a segment of foreign deoxyribonu-cleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a pro-tospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (cr-RNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to fre-quent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adap-tation. When a protospacer contains a spacer acqui-sition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phe-nomenon increases the repertoire of available spac-ers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation