Prespacers formed during primed adaptation associate with the Cas1–Cas2 adaptation complex and the Cas3 interference nuclease–helicase

Abstract

For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease–helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1–Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1–Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.

Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system

“…We cannot exclude that 3′-5′ exonucleases studied here (DnaQ, ExoT, RecBCD, and ExoVII) participate in this process, but their potential contribution may be masked by functional redundancy. According to our recent data (23), Cas3 remains associated with Cas1-Cas2 at least until the mature 33/37-nt prespacer is formed.…”

“…To reconstruct this process in vitro, we used a model DNA substrate based on a previously characterized dual forked substrate containing a 23-bp central duplex flanked by 5-nt 3′ and 5′ noncomplementary overhangs ( 22 ). To achieve stronger binding by Cas1-Cas2 and make the substrate more similar to prespacers observed in vivo ( 23 , 24 ), we introduced the 3′-TTC-5′ PAM and two additional nucleotides in the PAM-proximal 3′ overhang. To provide the recognition and processing of 5′ overhangs by RecJ ( 46 , 47 ), we extended both single-stranded 5′ ends to 19 nt ( Fig.…”

“…The 3′ ends are trimmed asymmetrically by unidentified nucleases such that a short 3′-NNTT-5′ overhang is left on the PAM-derived end. ( J ) Cas3 is transiently bound to Cas1-Cas2 and the 33/37-nt prespacer ( 23 ). ( K ) The Cas1-Cas2 complex integrates the prespacer into the CRISPR array.…”

“…Prespacers formed during primed adaptation have a double-stranded region of ~33 bp corresponding to a mature spacer plus four additional 3′-NNTT-5′ nucleotides on the PAM-derived end ( Fig. 1 ) ( 23 , 24 ). Therefore, in the present work, we will refer to asymmetrical 33/37-bp fragments or fragments of a very close size and structure containing the 3′-TTC-5′ motif within the PAM-derived 3′ end as prespacers.…”

Digging into the lesser-known aspects of CRISPR biology

“…However, it is unknown whether the Cas1-Cas2 complex captures ssDNA or dsDNA molecules, and it remains to be elucidated when and how the dsDNA spacers are generated from repair-derived ssDNA fragments. Concerning primed adaptation, it has been shown that the nuclease activity of Cas3, probably associated with the adaptation machinery in the so-called primed adaptation complex, produces prespacers in I-E and I-F systems (Shiriaeva et al 2020 ; Musharova et al 2021 ).…”