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Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion

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Abstract

Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides.

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References

  1. Pickar-Oliver A, Gersbach CA. The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Bio. 2019;20:490–507. https://doi.org/10.1038/s41580-019-0131-5.

    Article  CAS  Google Scholar 

  2. Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33:985–9. https://doi.org/10.1038/nbt.3290.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Moon SB, Kim DY, Ko J-H, Kim J-S, Kim Y-S. Improving CRISPR genome editing by engineering guide RNAs. Trends Biotechnol. 2019;37:870–81. https://doi.org/10.1016/j.tibtech.2019.01.009.

    Article  CAS  PubMed  Google Scholar 

  4. Basila M, Kelley ML, van Smith A, B. Minimal 2’-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. Plos One. 2017;12:e0188593. https://doi.org/10.1371/journal.pone.0188593.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020;5:1. https://doi.org/10.1038/s41392-019-0089-y.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ryan DE, Taussig D, Steinfeld I, Phadnis SM, Lunstad BD, Singh M, Vuong X, Okochi KD, McCaffrey R, Olesiak M, Roy S, Yung CW, Curry B, Sampson JR, Bruhn L, Dellinger DJ. Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res. 2018;46:792–803. https://doi.org/10.1093/nar/gkx1199.

    Article  CAS  PubMed  Google Scholar 

  7. Filippova J, Matveeva A, Zhuravlev E, Stepanov G. Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems. Biochimie. 2019;167:49–60. https://doi.org/10.1016/j.biochi.2019.09.003.

    Article  CAS  PubMed  Google Scholar 

  8. Alfonzo JD, Brown JA, Byers PH, Cheung VG, Maraia RJ, Ross RL. A call for direct sequencing of full-length RNAs to identify all modifications. Nat Genet. 2021;53:1113–6. https://doi.org/10.1038/s41588-021-00903-1.

    Article  CAS  PubMed  Google Scholar 

  9. Nakayama H, Yamauchi Y, Nobe Y, Sato K, Takahashi N, Shalev-Benami M, Isobe T, Taoka M. Method for direct mass-spectrometry-based identification of monomethylated RNA nucleoside positional isomers and its application to the analysis of Leishmania rRNA. Anal Chem. 2019;91:15634–43. https://doi.org/10.1021/acs.analchem.9b03735.

    Article  CAS  PubMed  Google Scholar 

  10. Sutton JM, Guimaraes GJ, Annavarapu V, van Dongen WD, Bartlett MG. Current state of oligonucleotide characterization using liquid chromatography–mass spectrometry: insight into critical issues. J Am Soc Mass Spectr. 2020;31:1775–82. https://doi.org/10.1021/jasms.0c00179.

    Article  CAS  Google Scholar 

  11. Kimura S, Dedon PC, Waldor MK. Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications. Nat Chem Biol. 2020;16:964–72. https://doi.org/10.1038/s41589-020-0558-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang J, Yan S, Chang L, Guo W, Wang Y, Wang Y, Zhang P, Chen H-Y, Huang S. Direct microRNA sequencing using nanopore-induced phase-shift sequencing. Iscience. 2020;23:100916. https://doi.org/10.1016/j.isci.2020.100916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wei B, Wang J, Cadang L, Goyon A, Chen B, Yang F, Zhang K (2022) Development of an ion pairing reversed-phase liquid chromatography-mass spectrometry method for characterization of clustered regularly interspaced short palindromic repeats guide ribonucleic acid. J Chromatogr A. 462839. https://doi.org/10.1016/j.chroma.2022.462839

  14. Kanavarioti A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Sci Rep-uk. 2019;9:1019. https://doi.org/10.1038/s41598-018-37642-z.

    Article  CAS  Google Scholar 

  15. Goyon A, Scott B, Kurita K, Crittenden CM, Shaw D, Lin A, Yehl P, Zhang K. Full sequencing of CRISPR/Cas9 single guide RNA (sgRNA) via parallel ribonuclease digestions and hydrophilic interaction liquid chromatography–high-resolution mass spectrometry analysis. Anal Chem. 2021;93:14792–801. https://doi.org/10.1021/acs.analchem.1c03533.

    Article  CAS  PubMed  Google Scholar 

  16. Goyon A, Scott B, Kurita K, Maschinot C, Meyer K, Yehl P, Zhang K. On-line sequencing of CRISPR guide RNAs and their impurities via the use of immobilized ribonuclease cartridges attached to a 2D/3D-LC–MS system. Anal Chem. 2021. https://doi.org/10.1021/acs.analchem.1c04350.

    Article  PubMed  Google Scholar 

  17. Tao J, Ningxi Y, Jaeah K, John-Ross M, Mildred K, Kanchana R, Edward JM, Vladimir P, Serenus H. Oligonucleotide sequence mapping of large therapeutic mRNAs via parallel ribonuclease digestions and LC-MS/MS. Anal Chem. 2019;91:8500–6. https://doi.org/10.1021/acs.analchem.9b01664.

    Article  CAS  Google Scholar 

  18. Paulines MJ, Wetzel C, Limbach PA. Using spectral matching to interpret LC-MS/MS data during RNA modification mapping. J Mass Spectrom. 2019;54:906–14. https://doi.org/10.1002/jms.4456.

    Article  CAS  PubMed  Google Scholar 

  19. Oberacher H, Pitterl F. On the use of ESI-QqTOF-MS/MS for the comparative sequencing of nucleic acids. Biopolym. 2009;91:401–9. https://doi.org/10.1002/bip.21156.

    Article  CAS  Google Scholar 

  20. Taucher M, Breuker K. Characterization of modified RNA by top-down mass spectrometry. Angewandte Chemie Int Ed Engl. 2012;51:11289–92. https://doi.org/10.1002/anie.201206232.

    Article  CAS  Google Scholar 

  21. Crittenden CM, Lanzillotti MB, Chen B. Top-down mass spectrometry of synthetic single guide ribonucleic acids enabled by facile sample clean-up. Anal Chem. 2023. https://doi.org/10.1021/acs.analchem.2c03030.

    Article  PubMed  Google Scholar 

  22. Hossain M, Limbach PA. Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products. RNA. 2007;13:295–303. https://doi.org/10.1261/rna.272507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wolf EJ, Grünberg S, Dai N, Chen T-H, Roy B, Yigit E, Corrêa IR. Human RNase 4 improves mRNA sequence characterization by LC–MS/MS. Nucleic Acids Res. 2022;50:e106–e106. https://doi.org/10.1093/nar/gkac632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vanhinsbergh CJ, Criscuolo A, Sutton JN, Murphy K, Williamson AJK, Cook K, Dickman MJ. Characterization and sequence mapping of large RNA and mRNA Therapeutics using mass spectrometry. Anal Chem. 2022;94:7339–49. https://doi.org/10.1021/acs.analchem.2c00765.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Edy VG, Szekely M, Loviny T, Dreyer C. Action of nucleases on double-stranded RNA. Eur J Biochem. 1976;61:563–72. https://doi.org/10.1111/j.1432-1033.1976.tb10051.x.

    Article  CAS  PubMed  Google Scholar 

  26. Loverix S, Winqvist A, Strömberg R, Steyaert J. Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. Chem Biol. 2000;7:651–8. https://doi.org/10.1016/s1074-5521(00)00005-3.

    Article  CAS  PubMed  Google Scholar 

  27. Huang M, Xu X, Qiu H, Li N. Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2021;1648:462184. https://doi.org/10.1016/j.chroma.2021.462184.

    Article  CAS  PubMed  Google Scholar 

  28. Birdsall RE, Gilar M, Shion H, Yu YQ, Chen W. Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis. Rapid Commun Mass Sp. 2016;30:1667–79. https://doi.org/10.1002/rcm.7596.

    Article  CAS  Google Scholar 

  29. Houser WM, Butterer A, Addepalli B, Limbach PA. Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry. Anal Biochem. 2015;478:52–8. https://doi.org/10.1016/j.ab.2015.03.016.

    Article  CAS  PubMed  Google Scholar 

  30. Shigematsu M, Kawamura T, Kirino Y. Generation of 2′,3′-cyclic phosphate-containing RNAs as a hidden layer of the transcriptome. Frontiers Gen. 2018;9:562. https://doi.org/10.3389/fgene.2018.00562.

    Article  CAS  Google Scholar 

  31. Honda S, Morichika K, Kirino Y. Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method. Nat Protoc. 2016;11:476–89. https://doi.org/10.1038/nprot.2016.025.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Schutz K, Hesselberth JR, Fields S. Capture and sequence analysis of RNAs with terminal 2′,3′-cyclic phosphates. RNA. 2010;16:621–31. https://doi.org/10.1261/rna.1934910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to thank Dr. Bifan Chen and Dr. Christopher M. Crittenden (Small Molecule Analytical Chemistry) from Genentech, Inc. for helpful discussions in optimization of mass spectrometry performance and analysis.

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  1. Department of Small Molecule Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA

    Steven Chin, Alexandre Goyon, Kelly Zhang & Kenji L. Kurita

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Correspondence to Kenji L. Kurita.

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Chin, S., Goyon, A., Zhang, K. et al. Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion. Anal Bioanal Chem 415, 2809–2818 (2023). https://doi.org/10.1007/s00216-023-04693-9

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