Biology subjects

Herring-Nicholas, A.

Publications and source records attributed to Herring-Nicholas, A..

3 recordsLinked to original sources

bioRxiv2025

TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs

RNA-guided CRISPR nuclease Cas9 cannot reliably differentiate between single nucleotide variations (SNVs) of targeted DNA sequences determined by their guide RNA (gRNA): they typically exhibit similar nuclease activities at any of those variations, unless the variation occurs with specific sequence contexts known as protospacer adjacent motifs (PAMs). Our approach, "TOP-SECRETS," generates gRNA variants that allow Cas9 ribonucleoproteins (RNPs) to reliably discriminate between healthy and disease-associated SNVs outside of PAMs.

bioengineering↗

bioRxiv2024

Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity

CRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target-specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably, in a spacer sequence-dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effectors activity and/or potential for off-target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, we present compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off-target combinations simultaneously in vitro for both CRISPR effector activity and specificity, by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water-in-oil microemulsions. With CCR, large numbers of the candidate gRNAs (output by computational design tools) can be immediately validated in parallel, and we show that CCR can be used to screen hundreds of thousands of extended gRNA (x-gRNAs) variants that can completely block cleavage at off-target sequences while maintaining high levels of on-target activity. We expect CCR can help to streamline the gRNA generation and validation processes for applications in biological and biomedical research.

bioengineering↗

bioRxiv2023

Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity

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.

bioengineering↗