Fast but not furious: a streamlined selection method for genome-edited cells

Abstract

In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods.

Identification of genome edited cells using CRISPRnano

“…DNA amplification was performed as previously described ( 4 ). Briefly, first-level PCR reactions were performed using 1 μl of lysate as a template in a 6.25 μl Phanta (Vazyme) PCR reaction according to the manufacturer's protocol (annealing temperature: 60°C; elongation time: 15 s, 19 cycles).…”

“…PCR products were pooled, size-separated using a 1% agarose gel and purified using GeneJET gel extraction kit (Thermo Fisher Scientific). Illumina library preparation was performed as previously described ( 4 ). Illumina libraries were sequenced using an Illumina MiSeq benchtop sequencer, a Nano V2 reagent kit (2 × 150) in a single-end sequencing setup (251 cycles).…”

“…DNA double-strand breaks (DSBs) induced by genome editing technologies are mainly repaired by two mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR). DSB induced by the NHEJ repair pathway leads to the formation of indels ( 3 , 4 ) that can result in frameshift mutations and often to loss-of-function phenotypes. Its counterpart, the HDR pathway, can be exploited to introduce the desired mutation, useful to model human disease.…”

Assessing the NLRP3 Inflammasome Activating Potential of a Large Panel of Micro- and Nanoplastics in THP-1 Cells

“…NLRP3 THP-1 mutant cells were generated as previously described [ 44 ]. Briefly, gRNAs were designed using the CRISPR design tool CHOPCHOP ( accessed on 12 June 2019) and cloned into a modified PX458 plasmid (Addgene #48138).…”

Investigating the Role of the NLRP3 Inflammasome Pathway in Acute Intestinal Inflammation: Use of THP-1 Knockout Cell Lines in an Advanced Triple Culture Model

“…CASP1 and NLRP3 THP-1 mutant cells were generated as previously described ( 26 ). Briefly, gRNAs were designed using the CRISPR design tool CHOPCHOP ( ) and cloned into a modified PX458 plasmid (Addgene #48138).…”