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

. 2022 Jun 22;12:10533. doi: 10.1038/s41598-022-14690-0

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

© The Author(s) 2022

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

Sample preparation: (A) A schematic illustration of the strategies for CRISPR/Cas9 mediated generation of constitutive gene knockouts in HCT116 cells. Upper panel: Single guide RNA targeting an early exon of a gene of interest will generate a double-strand break (DSB). In the presence of a DNA repair donor containing a promoterless gene trap (stop) cassette flanked with 5’ and 3’ in-frame homology arms (HA) two DSB repair outcomes are possible: (1) homology-directed repair (HDR) will result in an in-frame knockin (KI) of the stop cassette leading to gene inactivation or (2) the non-homologous end joining (NHEJ) repair will generate INDEL mutation (mut, represented by an asterisk), leading to a premature stop codon (red stop sign). Lower panel: targeting introns flanking an early critical exon with two guide RNAs will lead to an out-of-frame (OOF) deletion of the critical exon. Following the NHEJ-mediated repair, a premature stop codon (red stop sign) will be created in the downstream coding sequence, leading to gene knockout (KO). (B) KO cell lines were grown to densities of 20% and 80% confluency and samples were collected in triplicates.