Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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.

PMC Copyright notice

Figure 4. — Time-course analysis of the on/off-target cleavage of Cas12a using chimeric DNA–RNA guides on various target sequences. Target and off-target DNA cleavage experiments to confirm the target specificity of AsCpf1 using a chimeric DNA–RNA guide (DNA substitutions in the 3′-end) on (A) DNMT1, (B) GRIN2B, (C) HPRT1 and (D) RPL32P3 genes. Table: Information about on- and off-target sequences, Left: On-target cleavage over time, Middle: Off-target cleavage over time, Right: Target specificity (on/off cleavage ratio (%)) of AsCas12a using chimeric DNA–RNA guides (serial 4-nt and 8-nt DNA substitutions in the 3′-end of the (cr)RNA). NC: negative control, only Cas12a: only protein treated, WT: Wild-type crRNA was treated with Cas12a, +4 DNA: Chimeric crRNA (sequential 4-nt DNA substitution at 3′-end of crRNA) was treated with Cas12a, +8 DNA: Chimeric crRNA (sequential 8-nt DNA substitution at 3′-end of crRNA) was treated with Cas12a. All cleavage efficiencies were calculated from agarose gel separated band intensity (cleaved fragment intensity (%)/total fragment intensity (%)) for 60 min at 10 min interval points and normalized to wild-type (cr)RNA. 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). On- and off-target sequence information for each gene is shown in table at the top. The PAM sequence is underlined and shown in bold. Mismatch sequences to wild-type reference are shown in red.