title: CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Natalia
authors.initials: N
authors.lastname: Beloglazova
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kuznedelov
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Kirill A
authors.initials: KA
authors.lastname: Datsenko
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Ana
authors.initials: A
authors.lastname: Popovic
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Semenova
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia severik@waksman.rutgers.edu.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada a.iakounine@utoronto.ca.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D064130:CRISPR-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D004247:DNA / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA