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. 2020 Jul 20;48(15):8601–8616. doi: 10.1093/nar/gkaa605

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© The Author(s) 2020. 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 2. — Target DNA cleavage by Cas12a using a chimeric DNA–RNA guide in which the seed region of the (cr)RNA is replaced with DNA. (A) Comparative analysis of the target (DNMT1: orange) DNA cleavage efficiency using various (cr)RNAs harboring serial multiple or single DNA substitutions in the seed region close to the PAM (TTTN). (B) Comparative analysis of the target (CCR5: green) DNA cleavage efficiency. AsCas12a (cr)RNA was serially replaced with single DNA nucleotides from the PAM. The RNA portion of the (cr)RNA is shown in blue, and the DNA portion is shown in red (‘D’ indicates a DNA and the number of substituted DNA nucleotides is indicated). All cleavage efficiency were calculated from agarose gel separated band intensity (cleaved fragment intensity (%)/total fragment intensity (%)) and normalized to wild-type (cr)RNA (Figure S1). Data are shown as means ± s.e.m. from three independent experiments. P-values are calculated using a two-tailed Student's t-test (ns: not significant, *P< 0.05, **P< 0.01, ***P < 0.001, ****P < 0.0001).