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. 2019 Aug 8;47(16):8632–8648. doi: 10.1093/nar/gkz677

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Specific pre-spacer integration by Cas1–Cas2 into linear dsDNA targets. (A) Representation of the linear CRISPR target. Target consists of a leader sequence (yellow), repeat (black) and spacer (green). Leader-repeat (LR) and repeat–spacer junctions (RS) and points of integration (G1 and G36) are indicated. Palindromic inverted repeat sequences (IR 1 and IR 2) are marked. (B) Schematic of spacer integration in vitro with a minimal linear CRISPR target. Integration products include full-site integration, or half-site integrations at either the leader-repeat junction (LR) or repeat–spacer junction (RS). (C) In vitro spacer-integration assay with Cas1, Cas2, Csn2 and/or Cas9. Expected integration products corresponding to both junctions (LR or RS) are indicated. (D) High-throughput sequencing analysis of integration products represented as percent of total reads mapped throughout the linear CRISPR target.