Mutation detection in CRISPR experiments: Comparison of the Alt-R Genome Editing Detection Kit to targeted next generation sequencing
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.
Positive control: T7EI mutation cleavage assay
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.
Positive control: CRISPR editing (human, mouse, or rat HPRT)
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.
Negative control: CRISPR genome editing
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-Vas12a (Cpf1) System in human, mouse, and rat cells.