Strategies to detect and validate your CRISPR gene edit

Once you have carried out your gene editing experiment, how will you monitor the result? You’ve chosen a CRISPR strategy to introduce a gene edit into your target cells. How will you verify and characterize the edit? Depending on your experimental purpose and the nature of the gene edit, a variety of assays may be used, yielding various amounts and types of information.

This article summarizes the most commonly applied assays.

Method	Mutation type	Assay description	Application	Benefit	Limitation
A. Mismatch detection assay ^1,2	Insertion or deletion (INDEL)	Endonucleases such as T7 endonuclease I recognize structural deformities in DNA heteroduplexes. Cleavage fragments are separated by gel electrophoresis to determine DNA cleavage and gene editing %.	Rapid estimation of gene editing % in mixed populations. Identify most efficient experimental conditions. Screening single clones to further analyze.	Simple, cost effective.	No information on nucleotide sequence or functionality of targeted protein. Does not detect homozygous mutants. Not all mismatches are detected with same efficiency. Not amenable to high-thoughput experiments.
B. RFLP	Insertion or deletion (INDEL), single nucleotide polymorphisms (SNPs)	RFLP=Restriction Fragment Length Polymorphism. SNPs or INDELS that create or abolish restriction endonuclease recognition sites are amplified by locus-specific PCR primers. Following restriction digest, cleaved fragments are fractionated by gel electrophoresis into readily distinguishable patterns.	Rapid estimation of HDR % in mixed populations. Identify most efficient experimental conditions. Screening single clones to further analyze by sequencing.	Simple, cost effective. Detects SNPs and homozygous mutants.	Requires restriction site polymorphism. No information on nucleotide sequence or functionality.
C. Sanger sequencing	All	Genomic DNA surrounding putative mutation site is amplified by DNA sequencing.	Analyze the genotype of your single cell clones, such as allelic frequency and sequence of the edit.	Information on nucleotide sequence of each allele.	No functional information. Time consuming.
D. Western Blot	Insertion or deletion (INDEL), single nucleotide polymorphisms (SNPs), reporter genes	Cellular proteins are extracted and separated by polyacrylamide gel electrophoresis, then transferred to a membrane and hybridized to protein-specific antibodies, and secondary detection antibodies.	Monitor the protein expression level.	Simple, cost effective. Demonstrates likely protein knockout.	No information on nucleotide sequence. Absence/presence of protein detection does not always correlate with functional state. Requires specific antibodies.
E. Phenotypic assay	All	A variety of assays often specific to the target gene or cellular pathway, e.g. monitoring enzymatic activity, cell surface markers, apoptosis or fluorescent reporters.	Monitor or characterize phenotypic changes in the knockout cell lines and their biological relevance. Identify most efficient conditions. Select clones that show desired phenotype.	High throughput possible. Functional information. Biological relevance.	No information on nucleotide sequence.
F. NGS	All	Genomic DNA of clonal cell lines is sequenced in high throughput.	Analyze the genotype of your cells, such as allelic frequency and sequence of the edit. Identify off-targets. In context of a pooled CRISPR screen, identify enriched or depleted genes in a cell population.	High throughput, information on nucleotide sequence	No functional information.
G. TIDE³	Insertion or deletion (INDEL)	TIDE software employs the decomposition algorithm that quantifies identifies and frequencies of the predominant types of insertions and deletions in the DNA of a targeted cell population based on quantitative sequence trace data from two standard Sanger sequencing reactions of PCR amplicons of edited and control (unedited) samples.	Estimation of gene editing % in mixed populations and indel sizes.	Cost effective, gives an idea of spectrum of indels.	Doesn’t resolve large deletions, doesn’t work well with lower quality sequencing runs.

Summary

For most purposes, validation and characterization of edits on both the molecular and phenotypic level, will be required to assess their biological relevance.

For genomic screening purposes, a phenotypic assay may be a good starting point to rapidly screen gene edits that show a desired phenotype.

DNA mismatch assays, TIDE, RFLP, or phenotypic assays are often applied as starting points to assess the success of the CRISPR experiment and screen positive clones with desired knockout or knock-in mutations. Usually this first step, will be followed by more in-depth characterization of the nature of the edit on the sequence level, as well as on the functional level. Hence, in a typical gene editing experiment, a multitude of assays will be applied successively.