Bacteriophages suppress CRISPR–Cas immunity using RNA-based anti-CRISPRs

Many bacteria use CRISPR–Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR–Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR–Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR–Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR–Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6.


Prs217ACCGAGCUCGCATGCTTTCRFACCGCCGUGGCGTTAGTCGATTTGCTGCAAGGCGATTAAGTTGG
primer to clone pecto type I-F repeat-BsaI-repeat from pPF975 on pSC386 Prs215 AGCTCGGUGAATTCGAGC F primer to amplify backbone of pF178 to clone pecto type I-F repeat-BsaIrepeat into it, resulting in pSC386 Prs214 AGCTGCCUGTACGGCAGTGAACTGG AGAAACAGTAGAGAGTTGC R primer to amplify backbone of pF178 to clone pecto type I-F repeat-BsaIrepeat into it, resulting in pSC386 Prs220 ACTGCCGGAUAGGCAGCCAAGGAAT GAGACCTGCTG Primer replacing first repeat in pSC386 with SRU865, resulting in pSC387 Prs221 ATCCGGCAGUGAACTGGAGAAAC R Primer replacing first repeat in pSC386 with SRU865, resulting in pSC387 Ors132 GAAATGACACAGCCAACGCCCTGAA AATCGGCACAGG Oligo annealed with Ors134 for spacer ØTE insertion into pSC386, resulting in pSC391 Ors133 GGAATGACACAGCCAACGCCCTGAA AATCGGCACAGG Oligo annealed with Ors134 for spacer ØTE insertion into pSC387, resulting in pSC392 Ors134 TGAACCTGTGCCGATTTTCAGGGCG TTGGCTGTGTCA Oligo annealed with Ors132 or Ors133 for spacer ØTE insertion into pSC386 or pSC387 respectively, resulting in pSC391 and pSC392 respectively Prs31a ACTGTTTCUCCATCCGGGGCCTGCT CTC F primer for mamplification of RacrIF1 to clone behind PBAD +1 Prs78 ACTGTTTCUCCATTGGTGTTGCTGG ACTACCTG F primer for amplification of RacrIF1 to clone behind PBAD +1 amplification of RacrIF2 to clone behind PBAD +1 Prs80 ACTGTTTCUCCATTGGATTTGAACGG TTCACTGCCG F primer for amplification of RacrIF3 to clone behind PBAD +1 Prs65 ACTGTTTCUCCATCCGTGTTCCCCG CGTGTGC F primer for amplification of RacrIE1 to clone behind PBAD +1 Prs112 ACTGTTTCUCCATCCGCCCTCTCTGT CGTGGAAG F primer for amplification of RacrIE2 to clone behind PBAD +1 Prs55 ACTGTTTCUCCATTGGTCGCATCACA GCAAATAG F primer for amplification of RacrVA1 to clone behind PBAD +1 Prs54 ACTGTTTCUCCATTGTTTTTAAACCA TGTCAATTG F primer for amplification of RacrVA2 to clone behind PBAD +1 Prs107 ACTGTTTCUCCATTTTTTTAAAATGG TGAAAGTCTAAC F primer for amplification of RacrVA3 to clone behind PBAD +1 Scp14 AGTCCGAUCCCAACTATTTTGTCCG CCCAC F amplification of Racr candidates from twist fragments Prs108 ACCCAUGAGCACCATCATCGACCAG GAC F primer for amplification of AcrIC5 homologue and RacrIC1 behind PBAD and RBS Prs9 ACGGCCAGUTGATACGATTAGGACA ATGGTCACCGACG R amplification of Racr candidates from twist fragments Prs11 ACGGCCAGUTAGCCTGAGGGACTAA GGGAAACAGTTGTC R amplification of Racr candidates from twist fragments Prs83 ACGGCCAGUATCTACAAACAGTAGA AATTTAAAAAG R primer for amplification of RacrVA2 Prs111 ACGGCCAGUCGCCGTGCTTGTCAGA TGGTG R primer amplification of acr locus without RacrIC1 behind PBAD Prs120 Sanger sequencing cloning site pHerd-30T Prs212 AACGAAUCAGACAATTGACGG R primer to remove PBAD and AraC and clone RacrIF1 behind different promoters used on pPF2846 Prs213 AGCGUCGCCCGATTGCGC F primer to remove PBAD and AraC and clone RacrIF1 behind different promoters used on pPF2846 Ors124 AGCATAATCCCTAGGACTGAGCTAG CTATCAGAACGAAT Oligo annealed with Ors125 for insertion of promoter BBa_J23112 in front of RacrIF1 Ors125 CTGATAGCTAGCTCAGTCCTAGGGA TTATGCTAGCGT Oligo annealed with Ors124 for insertion of promoter BBa_J23112 in front of RacrIF1 Ors128 AGCATTGTACCTAGGACTGAGCTAG CCGTAAAAACGAAT Oligo annealed with Ors129 for insertion of promoter BBa_J23110 in front of RacrIF1 Ors129 TTTACGGCTAGCTCAGTCCTAGGTA CAATGCTAGCGT Oligo annealed with Ors128 for insertion of promoter BBa_J23110 in front of RacrIF1 Ors130 AGCACTGTACCTAGGACTGAGCTAG CCGTCAAAACGAAT Oligo annealed with Ors131 for insertion of promoter BBa_J23100 in front of RacrIF1 Ors131 TTGACGGCTAGCTCAGTCCTAGGTA CAGTGCTAGCGT Oligo annealed with Ors130 for insertion of promoter BBa_J23100 in front of RacrIF1 CRISPR array expansion PF174 CGTTAGAGTGATCGGGCTAC F primer for Pba CRISPR1 (binds in leader) PF175 CAATGGCTCAGGGGATTC R primer for Pba CRISPR1 (binds in spacer 2) PF176 GGTAACTACCGTAAAATAGGAACG F primer for Pba CRISPR2 (binds in leader) PF177 GCCTTTAAGCGCATGTCG R primer for Pba CRISPR2 (binds in spacer 2) PF178 CTTTAATAATCTGGTTGTTAGTGTG F primer for Pba CRISPR3 (binds in leader) PF179 CCTCAGAAAGCCGACTTC R primer for Pba CRISPR3 (binds in spacer 2) screening PF138 CACACTTTGCTATGCCATAG pPF781-derived plasmids, F primer cloning site PF1702 CGAAGACGAAAGGGCCTCGTGATAC GCAAGCTTTATGGCTTGTAAACCGTT TTGTG pPF781-derived plasmids, R primer cloning site PF2026 GGATCTCGACGCTCTCCCTT 6xHis-tag insertion in pPF2640, F primer PF757 GCTCTAGAGGGCCATGTTCCACAAA CAC 6xHis-tag insertion in pPF2640, R primer PF2084 TTGTACACGGCCGCATAATC pPF2640-derived plasmids, F primer cloning site PF1641 GCTAGTTATTGCTCAGCGG pPF2640-derived plasmids, R primer cloning site PF1218 GATGTCAAAACCGTCAAAGAG Pba gDNA contamination check, F primer PF1219 TTCTTGTACTGGTCGCGTTC Pba gDNA contamination check, R primer 5' RACE TSO GCTAATCATTGCAAGCAGTGGTATC AACGCAGAGTACATrGrGrG Template switching oligo used for 5' RACE (NEB) TSO-PCR CATTGCAAGCAGTGGTATCAAC F primer for amplification of 5' RACE product RT-RacrIF1 ATGCAGCTACGACCTATCCGGCAGT GAACGAGAGC reverse transcription primer binding in the RacrIF1 candidate sequence RT-PCR-RacrIF1 ACGAGAGCATCAGACAAGGCGGC R primer for amplification of 5' RACE product of RacrIF1 RT-crRNA ATGCAGCTACGACCTGTACGGCAGT GAACGCATCC reverse transcription primer binding in the crRNA RT-PCR-crRNA ACGCATCCGTCCAGACCGTATCG R primer for amplification of 5' RACE product of the crRNA RT-RacrIC1 ATGCAGCTACGATCGCGGGGCGCG ACCGCCG reverse transcription primer binding in the RacrIC1 candidate sequence RT-PCR-RacrIC1 GATCGCGGGGCGCGACCG R primer for amplification of 5' RACE product of RacrIC1 and for sanger sequencing of the PCR product PF861 TAATACGACTCACTATAGGG primer for sequencing of 5' RACE products cloned into pGEM-T Supplementary

Table 2 :
Strains and phages used in this study.

Table 3 :
Oligonucleotides used in this study.

Table 4 :
Plasmids used in this study.

Table 5 :
Overview of p-values retrieved from statistical analyses.
* note that the p-value is based on 1000 random samplings