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Nucleic Acids Res. 2014 May;42(9):5907-16. doi: 10.1093/nar/gku226. Epub 2014 Apr 11.

Pervasive generation of oppositely oriented spacers during CRISPR adaptation.

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Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia.
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia.
Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.
M.V. Lomonosov Moscow State University, Moscow, 119991, Russia.
Purdue University, West Lafayette, IN 47907, USA.
St Petersburg State Polytechnical University, St Petersburg, 195251, Russia


During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) 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 frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition 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 phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.

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