For detection of on-target or known off-target CRISPR events in cultured cells
The T7 endonuclease I (T7EI) mismatch cleavage assay detects on-target genome editing and estimates genome editing efficiency in CRISPR-treated cells.
Use the control primer mixes below with the Alt-R Genome Editing Detection Kit to determine editing efficiency in samples transfected with Alt-R CRISPR HPRT Positive Control crRNAs [available for the Alt-R CRISPR-Cas9 System and Alt-R CRISPR-Cas12a (Cpf1) System].
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 PCR design assistance, use the PrimerQuest® Tool at www.idtdna.com/PrimerQuest.
For both the Alt-R CRISPR-Cas9 System and the 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.
The Alt-R Genome Editing Detection Kit, a T7EI mismatch endonuclease assay, provides a good estimate of genome editing efficiency. However, because T7EI endonuclease does not recognize single-base insertions or deletions, this method underestimates editing efficiency when compared to next generation sequencing (NGS). The amount of underestimation by the T7EI assay varies by target and is dependent on the non-homologous end joining (NHEJ)-mediated repair events that follow Cas9 cleavage.
Figure 1. T7EI mismatch endonuclease assays provide a good estimate of genome editing efficiency, but underestimate efficiency when compared to next generation sequencing (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, genomic DNA samples 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 NGS (light blue bars). Amplicons were run an Illumina MiSeq® system 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.
Controls A and B from the Alt-R Genome Editing Detection Kit provide a robust control for the T7EI assay to show that the assay is functioning. Full-length PCR fragments for Controls A and B are 692 and 686 bp, respectively. T7EI digestion products are approximately 436 and 256 bp.
Figure 2. Easy-to-read results from T7EI mutation cleavage assay analyzed on a Fragment Analyzer™ system. PCR using template and primers in Controls A and B (Alt-R Genome Editing Detection Kit) were run using KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems). Cycling conditions were 5 min. 95°C; 30 x (20 sec. 98°C, 15 sec. 64°C, 30 sec. 72°C); 2 min. 72°C. PCR products were denatured and reannealed in a thermal cycler (10 min. 95°C; 95–85°C (ramp rate of –2°C/sec); 85–25°C (ramp rate of –0.3°C/sec). Sample 1 contains Control A PCR products, while Sample 2 contains homoduplexes and heteroduplexes of Control A and B PCR products. Reannealed PCR products were digested with 2 U of T7EI for 60 min at 37°C. Digestion reactions were analyzed on a Fragment Analyzer system (Advanced Analytical Technologies, Inc.). Trace (left) shows results from Sample 2. Gel image (right) shows results for Samples 1 and 2. LM = lower marker; UM = upper marker.
The results from positive control experiments are important for research publications and provide useful information, should you need to troubleshoot your experiments.
Confirm that your CRISPR editing conditions are working by using Alt-R HPRT Positive Control crRNAs (human, mouse, or rat). We offer distinct, positive control crRNAs for the Alt-R CRISPR-Cas9 and Alt-R CRISPR-Cas12a (Cpf1) Systems.
To monitor genome editing in your positive control samples, use the Alt-R Genome Editing Detection Kit and CRISPR Control Primer Mixes (human, mouse, or rat). The control PCR primers are designed to work with either Alt-R CRISPR-Cas9 or Alt-R CRISPR-Cas12a (Cpf1) Systems.
Figure 3. Sample data from T7EI digestion of Alt-R CRISPR HPRT Positive Controls. Genomic DNA from CRISPR-Cas9 (A) and CRISPR-Cpf1 (B) edited human, mouse, and rat HPRT controls were PCR amplified, digested using T7 endonuclease I, and run on the Fragment Analyzer system (Advanced Analytical Technologies, Inc.). Reference standard bands at 5000 bp (upper marker) and 35 bp (lower marker) are used to align the gel and analyze the results. Estimated band sizes for the cut and uncut positive control amplicons are listed in the tables. Cell lines used were HEK-293 (human), Hepa1-6 (mouse), and RG2 (rat). The PCR annealing temperature for human and mouse primers is 67°C, and for rat primers is 64°C.
Amplification of the negative control wells with your experimental primers and cycling conditions should result in only full-length products in the T7EI assay.
Using Alt-R CRISPR-Cas9 Negative Control crRNA #1, #2, or #3 in your editing experiments will support a conclusion that cleavage products from the T7EI assay result from target-specific recognition and cleavage by Cas9, and not other factors associated with transfection or electroporation. Note: Alt-R CRISPR-Cas9 Negative Control crRNA #1, #2, and #3 are computationally designed to not have homology to genomic targets in human, mouse, and rat.
IDT scientists have also computationally designed and tested negative control crRNA for the Alt-R CRISPR-Cas12a (Cpf1) System in human, mouse, and rat cells.