Pre ospacer 2 plasmid DNA protospacer 2 oligonucleotide DNA labeled strand omple

Question

Pre ospacer 2 plasmid DNA protospacer 2 oligonucleotide DNA labeled strand omplementary -non-complementary circular linearized plasmid-sp2 partner strand traerRNA 23 nt non-complementary stand bindng primer M PPM complementary strand binding primer asR Fig. 1. Cas9 is a DNA endonuclease guided by two RNA a spacer 2 sequence) programmed with a 42-nucleotide crRNA-sp2 (crRNA containing in the presence or absence of 75-nucleotide tracrRNA. The complex was added to or plasmid DNA bearing a sequence complementary to spacer 2 and a functional PAM. crRNA-sp1, specificity control M, DNA marker, kbp, hudeotiides 4 89). The complex was incubated with double- or single-stranded pair. S3A. crRNA-sp2 and tracrRNA DNAs harboring a sequence complementary to spacer 2 and a functional PAM (4). The complementary or noncomplementary strandsof the DNA were 5'-radiolabeled and annealed with a nonlabeled partner strand. nt, nucleotides. See fig, S3, Sequencing analysis of cleavage products from Fig. 1A. Termination of primer extension in the sequencing reaction indicates the position of the cleavage site. The 3' terminal A overhang (asterisks is an of the reaction. The products from Fig. 1B were analyzed alongside 5'end-labeled size markers derived from the complementary and noncomplementary strands of the target DNA duplex. M, complementarity to product. See fig. S5, representation crRNA-sp2, and protospacer DNA sequences. R (orange and the protospacer DNA represented. The PAM sequence is shown in gray; cleavage sites mapped in (C) and (D) are represented by blue arrows (C, red arrow [(D), complementary strand, and a red line [(D), noncomplementary strandi

Explanation / Answer

A] Cas9 is an endonuclease which is incorporated with a 42 nucleotide long crRNA, crRNA are RNAs that protect against invasive genetic materials . The crRNA contains a spacer 2 sequence . The spacers are non coding sequences between tandem repeats . tracrRNA is transactivating cr RNA which provide a stem loop structure and is ay or may not be programmed here. This complex was taken up by a linearized olasmid DNA ( plasmid dna is circular which has to be cut by specific endonycleases to form a linearized structure. The plasmid DNA comprises complementary sequence to the spacer 2 and PAM for active complementary base pairing.

B] The same complex now with tracrRNA was incubated with ssdna with complementary strands, the dna were radio labelled and were annealed with strands which are not labelled.

c] the cleavage products of the first complex is identified by identifying the location where primer is not extending anymore.3' terminal a overhangs are produced.

D] The cleavage products from the second complex were analyzed in accordance to the radio labelled markers .

E] The complete representation of all the sequence is shown in different colors. The complementary sites of crrnawhere to tracrrna shown. Photospacer shown in yellow color, PAM sequence in complementary and noncomplementary strands shown in grey color. Cleavage site of complementary and non complementary are strand shown by red line blue arrows.

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