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A simple method to detect on-target editing or measure genome editing efficiency in CRISPR experiments

The Alt-R® Genome Editing Detection Kit—a PCR-based, T7 endonuclease I (T7EI) assay

Applications and reagents for CRISPR genome editing are developing quickly. Whether you are using CRISPR-Cas9, CRISPR-Cpf1, or another CRISPR system, identifying on-target editing events is vital for interpreting your experimental results. We currently recommend the Alt-R Genome Editing Detection Kit to perform T7EI assays for CRISPR mutation detection (Figure 1). This method provides clean electrophoretic results, requires only standard molecular biology equipment, and does not usually require purification of PCR products prior to T7EI digestion.

Alt-R Genome Editing Detection Kit (T7EI assay)

Figure 1. Schematic of the T7EI assay, the basis of the Alt-R® Genome Editing Detection Kit. Use PCR to amplify the targeted genomic region (note: reagents for PCR are not part of the kit). Denature and reanneal PCR products in a thermal cycler to allow potential heteroduplex formation between wild-type and CRISPR–mutated DNA. Digest reannealed PCR products with T7EI, which cleaves mismatched DNA heteroduplexes. Analyze results using gel or capillary electrophoresis.

The Alt-R Genome Editing Detection Kit supplies the T7EI enzyme and buffer, as well as template-primer mixes for T7EI positive control reactions. You must design the target-specific PCR primers (see sidebar, Tips for using the Alt-R® Genome Editing Detection Kit). Use them with your preferred PCR reagents.

Specificity of T7EI for insertions or ≥2 base deletions 

T7EI recognizes insertions and deletions (indels) of ≥2 bases that are generated by non-homologous end joining (NHEJ) activity in CRISPR experiments. Because T7EI does not recognize 1 bp indels, T7EI assays can underrepresent total editing. The variance depends on the target and is affected by the type of NHEJ-mediated repair events that follow DNA cleavage (Figure 2). To enrich for successfully transfected cells, we offer a fluorescently labeled tracrRNA that enables fluorescence-activated cell sorting (FACS) (see sidebars, Tips for using the Alt-R™ Genome Editing Detection Kit and Additional resources).

Comparison of T7EI assay and NGS for detection of CRISPR editing

Figure 2. T7EI mismatch endonuclease assays provide a good estimate of genome editing efficiency, but underestimate efficiency when compared to NGS results. The Alt-R® CRISPR-Cas9 RNA oligonucleotides (30 nM) were introduced by lipofection into HEK-293 cells that stably express Streptococcus pyogenes Cas9. 3 PAM sites were targeted in each of 8 genes. To estimate editing efficiency, samples of genomic DNA from the transfected cells were tested using the Alt-R Genome Editing Detection Kit (dark blue bars), which provides reagents needed to run T7EI assays. The same DNA samples were also analyzed using targeted next generation sequencing (NGS) (light blue bars). Amplicons were run on a MiSeq® system (Illumina) and data were analyzed using a publicly available data processing program [Pinello L, Canver MC, et al. (2016) Nat Biotechnol 34:695–697.). Error bars represent standard deviation for triplicate lipofection experiments.

Note that in Alt-R CRISPR-Cpf1 experiments, our preliminary results suggest that comparable editing efficiencies are obtained using T7EI assays and NGS. Cpf1 endonuclease generates 5′ staggered cuts, and presumably, NHEJ repair results in fewer single base indels compared to NHEJ repair of the blunt-ended cuts created by Cas9 endonuclease.

For more information and protocols, visit the Alt-R Genome Editing Detection Kit web page.

Tips for using the Alt-R® Genome Editing Detection Kit

PCR design

Design PCR primers that amplify your experimental target site and adjacent sequences. We recommend using PCR amplicons that are 600–1000 bp in length with at least 100 bp flanking each side of the CRISPR cut site. Position the CRISPR cut site off-center to allow for 2 distinctive digestion product sizes on the gel.

Carefully optimize PCR conditions, and confirm by electrophoresis that a single PCR product is amplified from genomic DNA prior to editing.

For assistance with PCR assay design, use the PrimerQuest® Tool at www.idtdna.com/PrimerQuest.

Alt-R CRISPR-Cas9 System

Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550 is a fluorescently labeled tracrRNA that can be substituted for unlabeled tracrRNA in genome editing experiments using Cas9 nuclease. The fluorescent dye allows you to use fluorescence-activated cell sorting (FACS) analysis to concentrate transfected cells, which increases the likelihood of identifying cells with CRISPR-mediated alterations using the Alt-R Genome Editing Detection Kit.

Product focus

Alt-R® Genome Editing Detection Kit

If you want to estimate editing efficiency or detect on-target editing in your CRISPR experiments, we recommend using the Alt-R Genome Editing Detection Kit, a T7 endonuclease I–based assay. This kit is ideal for directly analyzing PCR products (i.e., no clean-up step required) to detect multiple-base insertions and deletions.

Learn more about the Alt-R Genome Editing Detection Kit.

Related products

  • Control PCR primers—for use with samples transfected with positive control crRNAs
  • For both Alt-R CRISPR-Cas9 System and Alt-R CRISPR-Cpf1 System, we offer recommended products and sequences for crRNA positive controls that target HPRT in human, mouse and rat cells. We have also designed and tested companion PCR primers for use with the Alt-R Genome Editing Detection Kit, which can be used to analyze editing efficiency in the positive control samples. These control PCR primer mixes can be used with either the Alt-R CRISPR-Cas9 System or the Alt-R CRISPR-Cpf1 System.

  • Alt-R CRISPR Systems
  • We offer 2 CRISPR systems based on delivery of ribonucleoproteins for genome editing. Each system has its own reagents. (Reagents have been optimized for use with each nuclease and are not interchangeable between systems.)

Additional resources

Alt-R® CRISPR-Cas9 System

CRISPR guide RNA format affects genome editing outcomes—Learn how use of different formats for the guide RNAs associated with CRISPR-Cas9 genome editing can lead to different editing outcomes. The optimized, short RNA oligos that make up the crRNA and tracrRNA components of the Alt-R CRISPR-Cas9 System outperform other CRISPR guide RNA formats. Unlike DNA expression constructs, short RNA oligos are unable to incorporate into the target genome, resulting in cleaner editing.

Use of fluorescently labeled Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550—Application note: Learn how fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP.

Getting started with Alt-R® CRISPR-Cas9 genome editing—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

Alt-R CRISPR-Cpf1 System

Cpf1 genome editing using the Alt-R® CRISPR-Cpf1 System—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cpf1 System, as well as the strengths and weaknesses of this powerful genome editing research tool.

Author: Maureen Young, PhD, is a senior scientific writer at IDT.

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