Detection of genome-edited mutant clones by a simple competition-based PCR method

Abstract

Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.

Mitochondria-specific photoactivation to monitor local sphingosine metabolism and function

“…There are two isoforms of sphingosine kinases in mammalian cells, sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2), both of which are capable of phosphorylating sphingosine into S1P ( Maceyka et al, 2005 ). To investigate the role of SphKs, we employed the CRISPR/Cas9-based genome editing strategy to generate a control and the double kinase knock-out cell line based on HeLa MZ cells( Figure 4—figure supplement 1 ) ( Ran et al, 2013 ; Liao et al, 2015 ; Harayama and Riezman, 2017 ). After showing that removing sphingosine kinases did not disrupt mitochondria morphology or membrane potential ( Figure 4—figure supplement 2 ), we carried out uncaging experiments using Mito-So in the cell lines and measured their sphingolipid levels.…”

“…Mutant cells were generated by the CRISPR/Cas9 system from Streptococcus pyogenes ( Ran et al, 2013 ), using the HPRT co-targeting strategy ( Liao et al, 2015 ) as previously described ( Harayama and Riezman, 2017 ). Target sequences (listed below) were selected based on high specificity and efficacy scores predicted by the CRISPOR algorithm ( Haeussler et al, 2016 ), and the corresponding pairs of oligo DNA were synthesized (Microsynth AG, Balgach, St. Gallen, Switzerland).…”

Detection of genome-edited cells by oligoribonucleotide interference-PCR

“…We demonstrated that genome-editing events at multiple loci can be detected in a single-tube endpoint ORNi-PCR (Fig. 7), which has not been achievable before using endpoint PCR-based methods 13 , 14 . In this regard, the primer combination should be more carefully considered to avoid overlap of amplicons in the endpoint PCR-based methods.…”

“…On the other hand, because ORNi-PCR generates positive PCR signals when one allele of the genome of a target cell is edited, it may be difficult to distinguish bi- and mono-allelic mutations by endpoint ORNi-PCR. This feature is one of the drawbacks of ORNi-PCR not found in endpoint PCR-based methods 13 , 14 (Table 1). Thus, endpoint ORNi-PCR should be primarily used to detect genome-edited cells possessing at least mono-allelic mutations at a target site before subsequent detection of mono- or bi-allelic mutations by other methods.…”

“…Although those methods are useful for detecting genome-edited cells, some require expensive equipment, such as real-time PCR machines or capillary gel electrophoresis systems, and they are also time-consuming. To address this issue, simpler methods using endpoint PCR were developed, 13 , 14 in which a primer designed against the target site is used in nearly the same way as in the real-time PCR-based method 9 . Endpoint PCR-based approaches can also be more cost-effective for the evaluation of indel mutations.…”