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Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis
CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In...
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Published in: | Analytical chemistry (Washington) 2021-11, Vol.93 (44), p.14792-14801 |
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creator | Goyon, Alexandre Scott, Brandon Kurita, Kenji Crittenden, Christopher M Shaw, David Lin, Andy Yehl, Peter Zhang, Kelly |
description | CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach. |
doi_str_mv | 10.1021/acs.analchem.1c03533 |
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Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. 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subjects | Biocompatibility Chemistry Chromatography Chromatography, Liquid CRISPR CRISPR-Cas Systems Digestion DNA sequencing Fragments Genomes High resolution Humans Hydrophilicity Hydrophobic and Hydrophilic Interactions Liquid chromatography Mass Spectrometry Mass spectroscopy Nuclease Optimization Ribonuclease T1 Ribonucleases Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems Scientific imaging Separation Spectroscopy |
title | Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis |
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