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Cell Rep. 2019 Sep 17;28(12):3157-3166.e4. doi: 10.1016/j.celrep.2019.08.033.

Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length.

Author information

1
Institute of Biotechnology, Vilnius University, Vilnius 10257, Lithuania.
2
Molecular Biophysics Group, Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Leipzig 04103, Germany.
3
HALOmem, Charles Tanford Protein Centre, Martin Luther University Halle-Wittenberg, Halle 06120, Germany.
4
Institute of Biotechnology, Vilnius University, Vilnius 10257, Lithuania. Electronic address: siksnys@ibt.lt.
5
Molecular Biophysics Group, Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Leipzig 04103, Germany. Electronic address: ralf.seidel@physik.uni-leipzig.de.

Abstract

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.

KEYWORDS:

CRISPR; Cascade; Cse1; Cse2; R-loop; Streptococcus thermophilus; crRNA; genome engineering; single molecule; type I-E

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