| Title |
Decision-making in cascade complexes harboring crRNAs of altered length / |
| Authors |
Songailienė, Inga ; Rutkauskas, Marius ; Šinkūnas, Tomas ; Manakova, Elena ; Wittig, Sabine ; Schmidt, Carla ; Šikšnys, Virginijus ; Seidel, Ralf |
| DOI |
10.1016/j.celrep.2019.08.033 |
| Full Text |
|
| Is Part of |
Cell reports.. Cambridge, MA : Cell Press. 2019, vol. 28, iss. 12, p. 3157-3166.. eISSN 2211-1247 |
| Keywords [eng] |
Cascade ; CRISPR ; crRNA ; Cse1 ; Cse2 ; genome engineering ; R-loop ; single molecule ; Streptococcus thermophilus ; type I-E |
| Abstract [eng] |
The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems. |
| Published |
Cambridge, MA : Cell Press |
| Type |
Journal article |
| Language |
English |
| Publication date |
2019 |
