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 (Table 1), together with examples that apply them (see below, application examples A-J).

Table 1.

Method

Mutation type

Assay description

Application

Benefit

Limitation

Mismatch detection assay 1,2
row1 mismatch

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 populationsA,E. Identify most efficient experimental conditionsB. Screening single clones to further analyzeD,E.

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.

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 populationsA,C. Identify most efficient experimental conditionsC. 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.

Sanger sequencing
row3 sanger

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 editE,G.

Information on nucleotide sequence of each allele.

No functional information. Time consuming.

Western Blot
row4 westernblot

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.

Monitor the protein expression levelA,E.

Demonstrates likely protein knockout.

No information on nucleotide sequence. Absence/presence of protein detection does not always correlate with functional state. Requires specific antibodies.

Phenotypic assay
row5 pheno

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 relevanceF, G,H,I,J. Identify most efficient conditionsF. Select clones that show desired phenotypeG,J.

High throughput possible. Functional information. Biological relevance.

No information on nucleotide sequence.

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 editI. 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.

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.

Application examples

Fluorescent Cas9 mRNA for enrichment ofCRISPR-mediated knockout and knock-in using synthetic guide RNA

  • A demonstration of the use of Edit-R Fluorescent Cas9 Nuclease mRNA and synthetic guide RNAs for enriching knockout and knock-in gene edited cells.

Optimization of reverse transfection of Dharmacon™ Edit-R™ synthetic crRNA and tracrRNA components with DharmaFECT™ transfection reagent in a Cas9-expressing cell line

  • Maximize success of your arrayed synthetic crRNA knockout screen with careful transfection optimization, editing efficiency and phenotypic readout.

Homology-directed repair with Dharmacon™ Edit-R™ CRISPR-Cas9 reagents and single-stranded DNA oligos

  • Create precise insertions using the homology-directed repair (HDR) machinery with a single-stranded DNA donor.

Microinjection of zebrafish embryos using Dharmacon™ Edit-R™ Cas9 Nuclease mRNA, synthetic crRNA, and tracrRNA for genome engineering

  • Successful gene editing in Zebrafish embryos, as confirmed by T7EI mismatch assay and fluorescent readout.

A CRISPR-Cas9 gene engineering workflow: generating functional knockouts using Dharmacon™ Edit-R™ Cas9 and synthetic crRNA and tracrRNA

  • Example of gene engineering workflow from delivery of CRISPR-Cas9 reagents to clonal cell isolation and characterization using the Edit-R gene engineering platform.

Optimized HDR-mediated fluorescent protein knock-in in K-562 cells using Edit-R™ CRISPR-Cas9 reagents and electroporation

  • A suggested protocol optimizing delivery of the HDR reagents in difficult to transfect K-652 cells using an electroporation method.

Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon™ Edit-R™ CRISPR-Cas9 reagents

  • Tagging the endogenous SEC61B gene using an EGFP donor plasmid.

Using machine learning to identify the best CRISPR-Cas9 targets for functional gene knockout

  • High-throughput phenotypic analysis of CRISPR functionality.

Identification of genes involved in cell cycle regulation using arrayed synthetic CRISPR RNA libraries in a multiparameter high-content assay

  • An arrayed synthetic crRNA screen targeting cell cycle regulation genes with multiparametric, high-content phenotypic analysis on the IN Cell Analyzer 2200.

High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries

  • An arrayed crRNA screen identifies genes involved in different phases of the cell cycle

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 (application examples B, H, I, J), 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 knockin 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 (See application examples above).

References

  1. R. D. Mashal, J. Koontz, J. Sklar, Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 9, 177-183 (1995).
  2. L. Vouillot, A. Thelie, N. Pollet, Comparison of T7EI and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5, 407-415 (2015).
  3. E. K. Brinkman, T. Chen, M. Amendola, B. van Steensel, Easy quantitative assessment of genome editing by sequence trace decomposition. NAR 42, Issue 22, Pages e168 (2014).

Author: Kathrin Kerschgens Ph.D. | Project Manager Cell Line Engineering

Additional Resources

CRISPR-Cas9 Gene Editing Applications

  • CRISPR-Cas9 systems can be used with custom RNA guides for several gene editing applications.

Resources - Gene Editing

  • Find product guides, FAQs and more.