Abstract
The hyperthermophilic archaeon Sulfolobus solfataricus carries an extensive array of clustered regularly interspaced short palindromic repeats (CRISPR) systems able to mediate DNA degradation of invading genetic elements when complementarity to the small CRISPR-derived (cr)RNAs is given. Studying virus defence in vivo with recombinant viral variants, we demonstrate here that an unexpectedly high number of mutations are tolerated between the CRISPR-derived guide RNAs (crRNAs) and their target sequences (protospacer). Up to 15 mismatches in the crRNA still led to ∼50% of DNA degradation, when these mutations were outside the ‘seed’ region. More than 15 mutations were necessary to fully abolished interference. Different from other CRISPR systems investigated in vivo, mutations outside the protospacer region indicated no need for a protospacer adjacent motif sequence to confer DNA interference. However, complementarity of only 3 nucleotides between the repeat-derived 5′ handle of the crRNA and nucleotides adjacent to the protospacer enabled self-recognition, i.e. protection of the host locus. Our findings show commonalities and differences among the various CRISPR-mediated defence systems and suggest that they should not merely be perceived as a ‘first-barrier-defence system’ but may be considered to have a broader mechanism that allows host cells to cope with viruses keeping them at reduced levels.
Highlights
A new defence mechanism that protects bacteria and archaea from invading genetic elements through an RNA-mediated DNA interference mechanism was discovered
Minimal requirement of sequence complementarity between CRISPR RNAs (crRNAs) and protospacer In our previous work, we have demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR) system in S. solfataricus can recognize and trigger degradation of a protospacer when up to four mutations are introduced into the protospacer region, and that the efficiency of transfection decreased with the increase of the number of mismatches between the spacer and protospacer [22]
Our study demonstrates the extraordinary ability of the S. solfataricus CRISPR-CRISPR-associated proteins (Cas) system in targeting protospacers with high numbers of mismatches to the complementary crRNA
Summary
A new defence mechanism that protects bacteria and archaea from invading genetic elements through an RNA-mediated DNA interference mechanism was discovered. After transcription of the CRISPR region, the full-length RNA is processed into small CRISPR RNAs (crRNAs) that mediate degradation of a complementary invading DNA with the help of protein complexes encoded adjacent to the CRISPR locus, called CRISPR-associated proteins (Cas) [1,2,3,4]. This defence mechanism is present in most archaea (87%) and in >45% of bacteria (CRISPRdb http://crispr.u-psud.fr/ crispr/) and shows a great diversity [5]. The authors identified the positions important in the protospacer adjacent sequence (PAS) that can block CRISPR activity when matching the central 4 nt of the 50 handle of the spacer, demonstrating how the
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