title: Analysis of CRISPR Pre-crRNA Cleavage.
abstract: We have examined the processing of precursor-clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (pre-crRNAs) of the Type I CRISPR-Cas system by incubation of radiolabeled model RNAs with recombinant CRISPR-associated (Cas) endoribonucleases, followed by denaturing polyacrylamide gel electrophoresis (PAGE) of the products. Determination of cleavage position is based on comparison with RNase T1 digestion and base hydrolysis products. The mechanism of cleavage is investigated by chemical and enzymatic characterization of the reaction products as well as by the demonstration that a specific 2'-deoxy substitution 5' to the scissile phosphate blocks endonucleolytic cleavage.
authors.affiliation: Department of Biochemistry, University of Alberta, 443 Medical Sciences Building, Edmonton, AB, Canada, T6G 2H7.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Garside
authors.forename: Andrew M
authors.initials: AM
authors.lastname: MacMillan
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D011232:Chemical Precipitation; D002725:Chloroform / Q000737:chemistry; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004722:Endoribonucleases / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry; D006868:Hydrolysis; D009691:Nucleic Acid Denaturation; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D059367:RNA Cleavage; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D012322:RNA Precursors; D019800:Phenol; D000431:Ethanol; D002725:Chloroform; D004722:Endoribonucleases