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Why do I need to test several crRNAs for functionality?

Effective HDR-mediated knockin requires high functionality and specificity, as well as very close proximity to the HDR insertion site. Our CRISPR RNA Configurator tool takes these factors into consideration and weights functionality and specificity scoring with proximity to the desired insertion site for each crRNA sequence. However, the highest ranking crRNAs for specificity and functionality may not always be in the optimal genomic location, increasing the importance of functionally testing several crRNAs.