(A) Cas9-tracrRNA:crRNA complexes were reconstituted using 42-nucleotide crRNA-sp2 and truncated tracrRNA constructs and assayed for cleavage activity as in Fig. (E) Schematic representation of tracrRNA, crRNA-sp2 and protospacer 2 DNA sequences regions of crRNA complementarity to tracrRNA (orange) and the protospacer DNA (yellow) are represented PAM sequence, grey cleavage sites mapped in (C) and (D) are represented by blue arrows (C), a red arrow (D, complementary strand) and a red line (D, non-complementary strand). 1B were analyzed alongside 5’ end-labeled size markers derived from the complementary and non-complementary strands of the target DNA duplex. The 3’ terminal A overhang (asterisk) is an artifact of the sequencing reaction. Termination of primer extension in the sequencing reaction indicates the position of the cleavage site. (C) Sequencing analysis of cleavage products from Fig. The complementary or non-complementary strands of the DNA were 5’-radiolabeled and annealed with a non-labeled partner strand. The complex was incubated with double- or single-stranded DNAs harboring a sequence complementary to spacer 2 and a functional PAM (4). (B) Cas9 was programmed with crRNA-sp2 and tracrRNA (nucleotides 4-89). crRNA-sp1, specificity control M, DNA marker refer to fig. The complex was added to circular or XhoI-linearized plasmid DNA bearing a sequence complementary to spacer 2 and a functional PAM. (A) Cas9 was programmed with a 42-nucleotide crRNA-sp2 (crRNA containing spacer 2 sequence) in the presence or absence of 75-nucleotide tracrRNA.
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