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bioRxiv · 10.1101/2023.01.11.523593

Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity

Herring-Nicholas, A.·Dimig, H.·Roesing, M.·Josephs, E. A.
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Abstract

For a CRISPR guide RNA (gRNA) with a specific target but activity at known "off-target" sequences, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5 nucleotide extensions near its DNA-targeting segment--a modification that can increase Cas9 gene editing specificity by orders of magnitude with certain 5- extension sequences, via some as-yet-unknown mechanism that makes de novo design of the extension sequence difficult to perform manually--to robustly identify extended gRNAs (x-gRNAs) that have been counter-selected against activity at those off-target sites and that exhibit significantly enhanced Cas9 specificity for their intended targets.

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Herring-Nicholas, A., Dimig, H., Roesing, M., Josephs, E. A.. 2023-01-12. Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity. https://doi.org/10.1101/2023.01.11.523593

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