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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 1. — S. thermophilus Cas1 and Cas2 accurately integrates pre-spacers in vitro and 10 bp of the leader sequence is essential for polarized integration. (A) Schematic of pre-spacer (PS) integration by Cas1–Cas2 into a plasmid target containing a minimal CRISPR array (pCRISPR). Integration of pre-spacers can occur as half-site intermediates at either junction of the repeat or as full-site products. (B) Integration assays with Cas1–Cas2 and radiolabeled pre-spacers visualized with ethidium bromide staining and autoradiography. Integration products corresponding to relaxed plasmids (R), unintegrated supercoiled plasmid (SC) and free pre-spacers (PS) are indicated. (C) Variants of the leader sequence mutations (L1, L2, L3) engineered on pCRISPR. Sites of spacer integration were identified by high-throughput sequencing and mapped to the plasmids on the plus (upper) and minus (lower) strands.