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

. 2021 Sep 30;11:19482. doi: 10.1038/s41598-021-98965-y

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

© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Cas9 D10A mediates efficient HDR distant from nick sites. (A) Ten HDR donor templates were designed with an EcoRI sequence positioned at varying distances (0-nt, 13-nt, 25-nt, 38-nt and 51-nt) from the left cleavage site of a paired-guide nickase design with a PAM-out orientation in HPRT1. ssODNs corresponding to the top and bottom (Btm) strand for each sequence were tested. Letters A–E indicate the position of the EcoRI insertion, the top strand ssODN is shown. (B) Cas9 D10A with gRNA pairs (left panel), or Cas9 WT with each of the individual gRNAs (middle and right panel) RNP complexes (Alt-R S.p. Cas9 D10A nickase or Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 crRNA and tracrRNA) were delivered at 4 µM (2 µM each RNP for nickase paired guides) along with 4 µM Alt-R Cas9 Electroporation Enhancer and 2 µM donor template by nucleofection to HEK293 cells (top) or K562 cells (bottom). HDR efficiency was evaluated by EcoRI cleavage of targeted amplicons. Data are represented as mean ± SEM of three biological replicates for D10A and two biological replicates for WT.