title: Substrate generation for endonucleases of CRISPR/cas systems.
abstract: The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.
authors.affiliation: Prokaryotic Small RNA Biology, Max-Planck-Institute for Terrestrial Microbiology.
authors.forename: Judith
authors.initials: J
authors.lastname: Zoephel
authors.forename: Srivatsa
authors.initials: S
authors.lastname: Dwarakanath
authors.forename: Hagen
authors.initials: H
authors.lastname: Richter
authors.forename: André
authors.initials: A
authors.lastname: Plagens
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
mesh_terms: D001483:Base Sequence; D048013:Acetivibrio thermocellus / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D012329:RNA, Bacterial; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases