Casa protospacer 2 mismatch mismatched targets Fig. 3. Cas9-catalyzed cleavage o

Question

Casa protospacer 2 mismatch mismatched targets Fig. 3. Cas9-catalyzed cleavage of target DNA requires an activating domain (right were cleaved in vitro byprogrammedCas9asin Fig. 1A (op left and used in tracrRNA and is governed by a seed sequence in the aRNA GAO for transformation assays of WT or preaRNA-deficient pyogenes (bottom left. nsformation efficiency was calculated as colonyforming units (CA) per aRNA complexes were reconstituted using 42 nudeotide crRNA-sp2 and trun- The tra constructs and were assayed for deavage adivity as in Fig. 18. (B) microgram of plasmid DNA Enror barsrepresent SDs for three biological replicates. cated tracrRNA cas9 programmed with fulllength tracrRNA and crRNA-sp2 truncations was as Plasmids containing and mutant protospacer2insets with varying extent of aRNA-target DNA mismatches (right were cieaved in vitro by programmed sayed for activity as in (A). (C Minimal regionsof tracrRNA and crRNA capable DNA deavage blue shaded region. (D Plasmids Cas90eft. The deavage reactions were further digested with Xmml. The 1880-and of guiding Cas9-mediated containing NT or mutant protospacer2sequences with indicated point mutations 800 bp fragments are Cas9-generated deavage products M DNA marker

Explanation / Answer

answers:

Summary: DNA interferences mechanism involving a dual- RNA structure that directs as Cas9 endonuclease to introduce site specific doublestrand break in target DNA. The tracrRNA:crRNA- guided Cas9 protein utilizes distinct endonuclease domains. HNH and RuvC-like, to cleave the two strands in target DNA. Target recognition by Cas9 requires both seed sequence in crRNA-binding region in DNA target.

1. Cas9 is a DNA endonuclease guided by two RNAs

2. Each Cas9 nuclease domain cleaves one DNA strand

3. Dual RNA requirements for target DNA binding and cleavage

4. A short sequences motif dictate R-loops formation

5. Cas9 can be progammed with a single chimeric RNA

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