Processing
Detecting CRISPR edits
Overview
CRISPR-dependent DNA alterations can sometimes be detected as phenotypic changes in cells. Also, the T7EI assay can be used as a quick assessment of genome editing success, and Sanger sequencing can determine on-target editing. However, the gold standard for detecting genome editing is next generation sequencing.
How to confirm CRISPR gene editing success
At the end of a CRISPR genome editing experimental workflow, it is important to determine if the intended edits were made and if there are unwanted edits not at the targeted location. The new genomic sequence at the target site can be detected using various methods.
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Everything you need to know about CRISPR, from A to Z, from theory to practice, for beginners as well as advanced users.
Phenotypic changes
The simplest but often the least specific method of detecting successful CRISPR genome editing is to observe phenotypes of edited cells. In some experiments, the expected phenotype from the gene editing process is known. For example, expression of a fluorescent protein may be turned on or off. In these cases, simply observing phenotypical changes will indicate if genome editing has been successful. However, if sequence information is needed, other assays must be performed as described below.
Enzymatic mismatch cleavage (gel-based) assays
Another simple method to detect on-target gene edits is to use enzymatic assays followed by gel analysis. Surveyor™ enzyme and T7EI are enzymes used for this purpose. T7EI is the more sensitive of the two and typically is the preferred choice of these methods. These methods do not show the actual sequence composition of the edited region but can indicate that a change in the genomic DNA sequence has occurred. Both assays are enzymatic mismatch cleavage methods and are available from IDT as the Surveyor Mutation Detection Kit and the Alt-R™ Genome Editing Detection Kit (the T7EI assay, Figure 1).
Figure 1. Schematic of T7EI assay.
For both kits, the workflows are as follows:
- Step 1—PCR amplification of control and CRISPR-edited genomic DNA. This step increases the amount of available DNA.
- Step 2—DNA duplex/heteroduplex formation. This step combines the original sequence with an edited sequence, resulting in a heteroduplex with mismatched bases at the edited location.
- Step 3—Treatment with nuclease. Depending on the kit, the nuclease is either Surveyor nuclease or T7 endonuclease I. The nuclease cuts mismatched DNA.
- Step 4—Analysis of DNA fragments. In this step, the digestion products are run on a gel. The mismatched DNA shows different sizes of bands compared to fully matched DNA.
Choosing between the Surveyor Kit and the Alt-R Genome Editing Detection Kit (T7EI assay)
The T7EI assay kit (Alt-R Genome Editing Detection Kit) is recommended for general use because it provides more sensitive detection of most genomic mutations. However, the T7EI method does not accurately detect single-nucleotide changes [1]. The Surveyor kit allows more reliable detection of single-nucleotide changes. However, neither method has the sensitivity or specificity of next generation sequencing (NGS).
Therefore, when using enzymatic mismatch cleavage for quick confirmation of edits, we recommend following up with NGS. NGS provides precise information about the nucleotide changes that have been generated by CRISPR genome editing and can be used to identify off-target effects as well; such detail is not possible with either enzymatic method.
Sequencing methods
Traditional Sanger sequencing was originally designed to detect a single nucleotide sequence, not mixed sequences in variably-edited samples. Therefore, Sanger sequencing is not practical for detection of CRISPR gene edits unless researchers either undertake prior separation of edited DNA (for example, separating cells into pure clones) or use specialized software for deconvolution of sequence data to identify several sequences at a single site. Both cloning and software approaches have been successful for use with Sanger sequencing, but like the enzymatic mismatch cleavage approach, no off-target data can be obtained except within the sequencing read.
Next generation sequencing
NGS is the recommended method for full investigation of CRISPR edits. Highly sensitive and specific, NGS allows detection of even small numbers of unintended edits at both the target site and at off-target sites. The standard approach recommended by IDT scientists is first to nominate off-target sites and then to use the rhAmpSeq™ CRISPR Analysis System to characterize on- and off-target editing. The system depends on IDT’s proprietary rhAmp PCR technology to generate amplicon libraries for targeted sequencing on Illumina® NGS platforms. The system also includes an advanced but accessible cloud-based data analysis pipeline (rhAmpSeq CRISPR Analysis Tool) for quantification of on- and off-target edits.