| Chapter title |
Chemical and Enzymatic Footprint Analyses of R-Loop Formation by Cascade-crRNA Complex
|
|---|---|
| Chapter number | 19 |
| Book title |
CRISPR
|
| Published in |
Methods in molecular biology, January 2015
|
| DOI | 10.1007/978-1-4939-2687-9_19 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-2686-2, 978-1-4939-2687-9
|
| Authors |
Ümit Pul |
| Editors |
Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran |
| Abstract |
Cascade-crRNA complexes mediate the identification of the invading foreign DNA and initiate its neutralization by formation of an R-loop (RNA-induced DNA-loop) at the crRNA-complementary sequence (protospacer). After initial unspecific binding to the double-stranded DNA, Cascade-crRNA complex slides along the DNA to find the protospacer. Once the target site is detected, the crRNA hybridizes to the complementary strand with subsequent displacement of the non-complementary strand to form an R-loop structure. Here, we describe how Cascade-DNA complexes and the Cascade-induced strand separation can be characterized in detail by combining chemical and enzymatic footprint analyses. Selective modification of unpaired thymines by permanganate (KMnO4) and the specific cleavage of single-stranded DNA by Nuclease P1 can be used to probe an R-loop formation by Cascade. Localization of the Cascade-crRNA complex on the DNA can be achieved by an Exonuclease III protection assay. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 2 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Professor > Associate Professor | 1 | 50% |
| Researcher | 1 | 50% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 2 | 100% |