Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Summary

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Abstract

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

Introduction

Reverse genetic approaches have allowed scientists to elucidate the effects of specific alterations in the genome on the cell or whole organism. For example, the expression of a particular gene can be attenuated by gene knockdown^1,2 (partial reduction) or gene knockout^3,4 (complete ablation) in order to determine the effect that this has on the function of the cell or on the development of the organism.

Gene knockout experiments have become easier since the introduction of sequence-specific programmable nucleases, such as zi....

Protocol

1. Obtaining CRISPR/Cas9-targeted Single-cell Clones

1. Seed HEPG2 cells on a 6-well plate at 500,000 cells per well in 2 mL of antibiotic-free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Incubate for 24 h at 37 °C and 5% CO₂.
2. Transfect cells with plasmid co-expressing Cas9 and specific sgRNA against the gene of interest using an appropriate transfection reagent as per the manufacturer's instructions.
   NOTE: For example, sgRNA can be cloned into the pSpCas9(BB)-2A-GFP vector, as described previously⁴.
3. Replace the culture medium 4 - 16 h after ....

Results

The fluorescent PCR-capillary gel electrophoresis technique described here is anticipated to be applicable to any targetable region in the genome in virtually any cell line that is amenable to foreign DNA delivery. We have previously demonstrated its application by targeting three genes in a colorectal cancer cell line¹². Here, we show its efficacy in genotyping a hepatocellular carcinoma cell line, HEPG2, targeted with a CRISPR/Cas9 construct against the Nucleosom.......

Discussion

The knocking out of a specific gene in a model cell line of choice has become routine for elucidating the role that the gene plays in that particular cellular context. In fact, several genome-wide screens are currently available that use the CRISPR/Cas9 system to target virtually all known human genes in the genome^14,15,16. With these large-scale screens (or even small-scale targeting of individual genes of interest), it is impo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPG2 cells	ATCC	HB-8065
HyClone Dulbecco's Modified Eagles Medium (DMEM)	Thermo Fisher Scientific	SH30022.01
HyClone Fetal Bovine Serum	Thermo Fisher Scientific	SV30160.03
pSpCas9(BB)-2A-GFP plasmid	Addgene	PX458
Lipofectamine 2000	Thermo Fisher Scientific	11668027
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056
Trypsin-EDTA (0.5%), no phenol red	Thermo Fisher Scientific	15400054
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
HyClone Water, Molecular Biology Grade	GE Healthcare	SH30538.02
CRISPR sgRNA insert oligonucleotide (sense)	AITbiotech	None	Sequence: 5'-CACCGCTAACCTTTCAGCCTGCCTA-3'
CRISPR sgRNA insert oligonucleotide (anti-sense)	AITbiotech	None	Sequence: 5'-AAACTAGGCAGGCTGAAAGGTTAGC-3'
Unlabeled PCR amplification forward primer	AITbiotech	None	Sequence: 5'-CACTAACTCCAATGCTTCAGTTTC-3'; this primer is also used to sequence PCR amplified alleles
6-FAM-labeled fluorescent PCR forward primer	AITbiotech	None	Sequence: 5'-6-FAM-CACTAACTCCAATGCTTCAGTTTC-3'
HEX-labeled fluorescent PCR forward primer	AITbiotech	None	Sequence: 5'-HEX-CACTAACTCCAATGCTTCAGTTTC-3'
Unlabeled PCR reverse primer	AITbiotech	None	Sequence: 5'-CCTCTTCCAAGTCTGCTTATGT-3'
Taq PCR Core Kit	QIAGEN	201223
Hi-Di Formamide	Thermo Fisher Scientific	4311320
GeneScan 500 LIZ Dye Size Standard	Thermo Fisher Scientific	4322682
MicroAmp Optical 96-Well Reaction Plate	Thermo Fisher Scientific	4306737
3500xL Genetic Analyzer	Thermo Fisher Scientific	4405633
3500 Series 2 program	Thermo Fisher Scientific	4476988
Gene Mapper 5 program	Thermo Fisher Scientific	4475073
Gentra Puregene Cell Kit	QIAGEN	1045696
Wizard SV Gel and PCR Clean-Up System	Promega	A9282
NAP1L1 Antibody (N-term)	Abgent	AP1920b
Nuclear Matrix Protein p84 antibody [5E10]	GeneTex	GTX70220
Peroxidase AffiniPure Goat Anti-Rabbit IgG	Jackson ImmunoResearch	111-035-144
Peroxidase AffiniPure Sheep Anti-Mouse IgG	Jackson ImmunoResearch	515-035-003