Use of CRISPR-Cas9 has revolutionized targeted genome editing. The process requires the Cas9 enzyme and guide RNA (gRNA: tracrRNA complexed with crRNA). The crRNA contains a variable region, which directs the Cas9 enzyme to the target DNA location. Designing a potent and specific gRNA is essential for successful genome editing. Yet, designing high-quality gRNA sequences remains a challenge.
- A well-designed gRNA should be potent. We and others demonstrated that the potency of gRNAs varies dramatically, even within the same target locus. Thus, choosing a gRNA with high on-target efficacy can increase the success of your CRISPR experiments.
- A well-designed gRNA should also be specific to the target. Specific gRNAs have low off-target activity. The CRISPR-Cas9 enzyme can tolerate up to four mismatches between the gRNA and its target, and one nucleotide gap. When tolerable mismatches between the gRNA and the target are present, cleavage can occur. Our studies show that a significant portion of off-target activity occurs within gene coding sequences. Therefore, the off-target activity can lead to inaccurate interpretation of the CRISPR experimental results. In addition, high off-target activity can cause depletion of the Cas9 enzyme or even cell death.
With those crucial elements in mind, we developed a gRNA design tool. We implemented a machine learning algorithm for designing high-quality gRNA sequences with high on-target and low off-target activity in 5 commonly studied species (human, mouse, rat, zebrafish, and nematode). Our tools also design gRNA sequences against custom targets, and can check your existing gRNA designs for potency and specificity.
In this webinar, Dr Matthew McNeill, a staff scientist in the bioinformatics group at IDT, gives a brief introduction to CRISPR-Cas9 genome editing and reviews the data generated by IDT scientists to illustrate the gRNA design tool’s performance. He also provides a demonstration of the Alt-R CRISPR-Cas9 gRNA design tool’s use in predicting and designing ideal gRNAs for genome editing experiments.