Loading…

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...

Full description

Saved in:
Bibliographic Details
Published in:Analytical chemistry (Washington) 2021-11, Vol.93 (44), p.14792-14801
Main Authors: Goyon, Alexandre, Scott, Brandon, Kurita, Kenji, Crittenden, Christopher M, Shaw, David, Lin, Andy, Yehl, Peter, Zhang, Kelly
Format: Article
Language:English
Subjects:
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by cdi_FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3
cites cdi_FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3
container_end_page 14801
container_issue 44
container_start_page 14792
container_title Analytical chemistry (Washington)
container_volume 93
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
format article
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_2587006102</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2600348443</sourcerecordid><originalsourceid>FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3</originalsourceid><addsrcrecordid>eNp9kctu1DAUhi0EokPhDRCyxKYsMvUtt-UopZ2RBqgysI4c20lceeLUTpCy4x14nL5NnwQPM-2CBasj2d_5fY4_AN5jtMSI4Esu_JL33IhO7ZdYIBpT-gIscExQlGQZeQkWCCEakRShM_DG-zuEMEY4eQ3OKEvyHKd0AR6uJ2PgTt1Pqhe6b6FtYFFudrflZcF9DnfhzCh4M2mpYPl1BS98G8on-FNzeMsdN0YZWOra9pMwinsFr3Sr_Kht7yHvJVzP0tmh00YLuOlH5bg4XMKtvg-hsOic3fPRto4P3fz46_dat11UKm_N9Jf7wr2Hu0GJMYBqdDNcha1nr_1b8Krhxqt3p3oOflx__l6so-23m02x2kacETJGYdM6ZiTPYpkQltaCsjhLBZMsFjlVDcV13iiWojTNEplmKSYCcykoFkTKhNNzcHHMHZwN_-THaq-9UMbwXtnJVyTEIZQEKwH9-A96ZycX5g1UEmywjDEaKHakhLPeO9VUg9N77uYKo-ogtwpyqye51UluaPtwCp_qvZLPTU82A4COwKH9-eH_Zv4BrpO2Bw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2600348443</pqid></control><display><type>article</type><title>Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis</title><source>American Chemical Society:Jisc Collections:American Chemical Society Read &amp; Publish Agreement 2022-2024 (Reading list)</source><creator>Goyon, Alexandre ; Scott, Brandon ; Kurita, Kenji ; Crittenden, Christopher M ; Shaw, David ; Lin, Andy ; Yehl, Peter ; Zhang, Kelly</creator><creatorcontrib>Goyon, Alexandre ; Scott, Brandon ; Kurita, Kenji ; Crittenden, Christopher M ; Shaw, David ; Lin, Andy ; Yehl, Peter ; Zhang, Kelly</creatorcontrib><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.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/acs.analchem.1c03533</identifier><identifier>PMID: 34699173</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>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</subject><ispartof>Analytical chemistry (Washington), 2021-11, Vol.93 (44), p.14792-14801</ispartof><rights>2021 The Authors. Published by American Chemical Society</rights><rights>Copyright American Chemical Society Nov 9, 2021</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3</citedby><cites>FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3</cites><orcidid>0000-0001-6515-544X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34699173$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Goyon, Alexandre</creatorcontrib><creatorcontrib>Scott, Brandon</creatorcontrib><creatorcontrib>Kurita, Kenji</creatorcontrib><creatorcontrib>Crittenden, Christopher M</creatorcontrib><creatorcontrib>Shaw, David</creatorcontrib><creatorcontrib>Lin, Andy</creatorcontrib><creatorcontrib>Yehl, Peter</creatorcontrib><creatorcontrib>Zhang, Kelly</creatorcontrib><title>Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><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.</description><subject>Biocompatibility</subject><subject>Chemistry</subject><subject>Chromatography</subject><subject>Chromatography, Liquid</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Digestion</subject><subject>DNA sequencing</subject><subject>Fragments</subject><subject>Genomes</subject><subject>High resolution</subject><subject>Humans</subject><subject>Hydrophilicity</subject><subject>Hydrophobic and Hydrophilic Interactions</subject><subject>Liquid chromatography</subject><subject>Mass Spectrometry</subject><subject>Mass spectroscopy</subject><subject>Nuclease</subject><subject>Optimization</subject><subject>Ribonuclease T1</subject><subject>Ribonucleases</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Guide, CRISPR-Cas Systems</subject><subject>Scientific imaging</subject><subject>Separation</subject><subject>Spectroscopy</subject><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><recordid>eNp9kctu1DAUhi0EokPhDRCyxKYsMvUtt-UopZ2RBqgysI4c20lceeLUTpCy4x14nL5NnwQPM-2CBasj2d_5fY4_AN5jtMSI4Esu_JL33IhO7ZdYIBpT-gIscExQlGQZeQkWCCEakRShM_DG-zuEMEY4eQ3OKEvyHKd0AR6uJ2PgTt1Pqhe6b6FtYFFudrflZcF9DnfhzCh4M2mpYPl1BS98G8on-FNzeMsdN0YZWOra9pMwinsFr3Sr_Kht7yHvJVzP0tmh00YLuOlH5bg4XMKtvg-hsOic3fPRto4P3fz46_dat11UKm_N9Jf7wr2Hu0GJMYBqdDNcha1nr_1b8Krhxqt3p3oOflx__l6so-23m02x2kacETJGYdM6ZiTPYpkQltaCsjhLBZMsFjlVDcV13iiWojTNEplmKSYCcykoFkTKhNNzcHHMHZwN_-THaq-9UMbwXtnJVyTEIZQEKwH9-A96ZycX5g1UEmywjDEaKHakhLPeO9VUg9N77uYKo-ogtwpyqye51UluaPtwCp_qvZLPTU82A4COwKH9-eH_Zv4BrpO2Bw</recordid><startdate>20211109</startdate><enddate>20211109</enddate><creator>Goyon, Alexandre</creator><creator>Scott, Brandon</creator><creator>Kurita, Kenji</creator><creator>Crittenden, Christopher M</creator><creator>Shaw, David</creator><creator>Lin, Andy</creator><creator>Yehl, Peter</creator><creator>Zhang, Kelly</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7TA</scope><scope>7TB</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-6515-544X</orcidid></search><sort><creationdate>20211109</creationdate><title>Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis</title><author>Goyon, Alexandre ; Scott, Brandon ; Kurita, Kenji ; Crittenden, Christopher M ; Shaw, David ; Lin, Andy ; Yehl, Peter ; Zhang, Kelly</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Biocompatibility</topic><topic>Chemistry</topic><topic>Chromatography</topic><topic>Chromatography, Liquid</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Digestion</topic><topic>DNA sequencing</topic><topic>Fragments</topic><topic>Genomes</topic><topic>High resolution</topic><topic>Humans</topic><topic>Hydrophilicity</topic><topic>Hydrophobic and Hydrophilic Interactions</topic><topic>Liquid chromatography</topic><topic>Mass Spectrometry</topic><topic>Mass spectroscopy</topic><topic>Nuclease</topic><topic>Optimization</topic><topic>Ribonuclease T1</topic><topic>Ribonucleases</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Guide, CRISPR-Cas Systems</topic><topic>Scientific imaging</topic><topic>Separation</topic><topic>Spectroscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Goyon, Alexandre</creatorcontrib><creatorcontrib>Scott, Brandon</creatorcontrib><creatorcontrib>Kurita, Kenji</creatorcontrib><creatorcontrib>Crittenden, Christopher M</creatorcontrib><creatorcontrib>Shaw, David</creatorcontrib><creatorcontrib>Lin, Andy</creatorcontrib><creatorcontrib>Yehl, Peter</creatorcontrib><creatorcontrib>Zhang, Kelly</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics &amp; Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical &amp; Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology &amp; Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts – Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Goyon, Alexandre</au><au>Scott, Brandon</au><au>Kurita, Kenji</au><au>Crittenden, Christopher M</au><au>Shaw, David</au><au>Lin, Andy</au><au>Yehl, Peter</au><au>Zhang, Kelly</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography–High-Resolution Mass Spectrometry Analysis</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2021-11-09</date><risdate>2021</risdate><volume>93</volume><issue>44</issue><spage>14792</spage><epage>14801</epage><pages>14792-14801</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><abstract>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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>34699173</pmid><doi>10.1021/acs.analchem.1c03533</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0001-6515-544X</orcidid><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 0003-2700
ispartof Analytical chemistry (Washington), 2021-11, Vol.93 (44), p.14792-14801
issn 0003-2700
1520-6882
language eng
recordid cdi_proquest_miscellaneous_2587006102
source American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list)
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
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-24T23%3A02%3A39IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Full%20Sequencing%20of%20CRISPR/Cas9%20Single%20Guide%20RNA%20(sgRNA)%20via%20Parallel%20Ribonuclease%20Digestions%20and%20Hydrophilic%20Interaction%20Liquid%20Chromatography%E2%80%93High-Resolution%20Mass%20Spectrometry%20Analysis&rft.jtitle=Analytical%20chemistry%20(Washington)&rft.au=Goyon,%20Alexandre&rft.date=2021-11-09&rft.volume=93&rft.issue=44&rft.spage=14792&rft.epage=14801&rft.pages=14792-14801&rft.issn=0003-2700&rft.eissn=1520-6882&rft_id=info:doi/10.1021/acs.analchem.1c03533&rft_dat=%3Cproquest_cross%3E2600348443%3C/proquest_cross%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-a422t-991b542985d6247bc34587c4d45c93ef31b9fe4707786d78712c1adc31c2dd6a3%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=2600348443&rft_id=info:pmid/34699173&rfr_iscdi=true