Type II CRISPR-Cas System Nucleases: a Pipeline for Prediction and  in vitro  Characterization

А. A. Vasileva; Васильева А. А.; S. A. Aliukas; Алюкас С. А.; P. A. Selkova; Селькова П. А.; A. N. Arseniev; Арсениев А. Н.; V. E. Chernova; Чернова В. Е.; O. S. Musharova; Мушарова О. С.; E. I. Klimuk; Климук Е. И.; M. A. Khodorkovskii; Ходорковский М. А.; K. V. Severinov; Северинов К. В.

doi:10.31857/S0026898423030163

Type II CRISPR-Cas System Nucleases: a Pipeline for Prediction and in vitro Characterization

Autores: Vasileva А.A.¹^,2, Aliukas S.A.³, Selkova P.A.¹^,2, Arseniev A.N.¹^,2, Chernova V.E.², Musharova O.S.¹, Klimuk E.I.¹, Khodorkovskii M.A.², Severinov K.V.¹^,4
Afiliações:
1. Institute of Molecular Genetics, Russian Academy of Sciences
2. Peter the Great St. Petersburg Polytechnic University
3. LLC “Biotech campus”
4. Waksman Institute of Microbiology
Edição: Volume 57, Nº 3 (2023)
Páginas: 546-560
Seção: МЕТОДЫ
URL: https://journals.rcsi.science/0026-8984/article/view/138647
DOI: https://doi.org/10.31857/S0026898423030163
EDN: https://elibrary.ru/CHWIKZ
ID: 138647

Citar

Texto integral

Acesso aberto
Acesso é fechado

Acesso está concedido
Acesso é fechado

Somente assinantes

Resumo
Sobre autores
Bibliografia
Arquivos suplementares
Estatísticas

Resumo

The use of CRISPR-Cas bacterial adaptive immunity systems components for targeted DNA changing has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editor-s are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-strand breaks into DNA regions complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an actual task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning and isolation of recombinant Cas9 proteins, testing for nuclease activity in vitro, and determining the PAM sequence required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

Palavras-chave

CRISPR, Cas9, nuclease, genome editing, bioinformatics search

Bibliografia

Jansen R., van Embden J.D.A., Gaastra W., Schouls L.M. (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565‒1575.
Barrangou R., Fremaux C., Deveau H., Richards M., Boyaval P., Moineau S., Romero D.A., Horvath P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science. 315, 1709‒1712.
Marraffini L.A., Sontheimer E.J. (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 322, 1843‒1845.
Garneau J.E., Dupuis M.-È., Villion M., Romero D.A., Barrangou R., Boyaval P., Fremaux C., Horvath P., Magadán A.H., Moineau S. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 468, 67‒71.
Makarova K.S., Wolf Y.I., Iranzo J., Shmakov S.A., Alkhnbashi O.S., Brouns S.J.J., Charpentier E., Cheng D., Haft D.H., Horvath P., Moineau S., Mojica F.J.M., Scott D., Shah S.A., Siksnys V., Terns M.P., Venclovas Č., White M.F., Yakunin A.F., Yan W., Zhang F., Garrett R.A., Backofen R., van der Oost J., Barrangou R., Koonin E.V. (2020) Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67‒83.
Cong L., Ran F.A., Cox D., Lin S., Barretto R., Habib N., Hsu P.D., Wu X., Jiang W., Marraffini L.A., Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819‒823.
Jinek M., East A., Cheng A., Lin S., Ma E., Doudna J. (2013) RNA-programmed genome editing in human cells. Elife. 2, e00471.
Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013) RNA-guided human genome engineering via Cas9. Science. 339, 823‒826.
Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471, 602–607.
Shah S.A., Erdmann S., Mojica F.J.M., Garrett R.A. (2013) Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899.
Luscombe N.M., Laskowski R.A., Thornton J.M. (2001) Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Res. 29, 2860–2874.
Chatterjee P., Lee J., Nip L., Koseki S.R.T., Tysinger E., Sontheimer E.J., Jacobson J.M., Jakimo N. (2020) A Cas9 with PAM recognition for adenine dinucleotide. Nat. Commun. 11, 2474.
Wei J., Hou L., Liu J., Wang Z., Gao S., Qi T., Gao S., Sun S., Wang Y. (2022) Closely related type II-C Cas9 orthologs recognize diverse PAMs. Elife. 11, e77825.
Fedorova I., Vasileva A., Selkova P., Abramova M., Arseniev A., Pobegalov G., Kazalov M., Musharova O., Goryanin I., Artamonova D., Zyubko T., Shmakov S., Artamonova T., Khodorkovskii M., Severinov K. (2020) PpCas9 from Pasteurella pneumotropica ‒ a compact type II-C Cas9 ortholog active in human cells. Nucleic Acids Res. 48, 12297–12309.
Bland C., Ramsey T.L., Sabree F., Lowe M., Brown K., Kyrpides N.C., Hugenholtz P. (2007) CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics. 8, 209.
Couvin D., Bernheim A., Toffano-Nioche C., Touchon M., Michalik J., Néron B., Rocha E.P.C., Vergnaud G., Gautheret D., Pourcel C. (2018) CRI-SPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 46, W246–W251.
Edgar R.C. (2007) PILER-CR: fast and accurate identification of CRISPR repeats. BMC Bioinformatics. 8, 18.
Biswas A., Staals R.H.J., Morales S.E., Fineran P.C., Brown C.M. (2016) CRISPRDetect: a flexible algorithm to define CRISPR arrays. BMC Genomics. 17, i356–i356.
Mitrofanov A., Alkhnbashi O.S., Shmakov S.A., Makarova K.S., Koonin E.V., Backofen R. (2021) CRISPRidentify: identification of CRISPR arrays using machine learning approach. Nucleic Acids Res. 49, e20.
Mistry J., Finn R.D., Eddy S.R., Bateman A., Punta M. (2013) Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic A-cids Res. 41, e121.
Dooley S.K., Baken E.K., Moss W.N., Howe A., Young J.K. (2021) Identification and evolution of Cas9 tracrRNAs. CRISPR J. 4, 438–447.
Mitrofanov A., Ziemann M., Alkhnbashi O.S., Hess W.R., Backofen R. (2021) CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection. Bioinformatics. 38, ii42–ii48.
Livny J., Waldor M.K. (2007) Identification of small RNAs in diverse bacterial species. Curr. Opin. Microbiol. 10, 96–101.
Lorenz R., Bernhart S.H., zu Siederdissen C.H., Tafer H., Flamm C., Stadler P.F., Hofacker I.L. (2011) ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26.
Harrington L.B., Burstein D., Chen J.S., Paez-Espino D., Ma E., Witte I.P., Cofsky J.C., Kyrpides N.C., Banfield J.F., Doudna J.A. (2019) Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 362, 839–842.
Zhang Y., Heidrich N., Ampattu B.J., Gunderson C.W., Seifert H.S., Schoen C., Vogel J., Sontheimer E.J. (2013) Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell. 50, 488–503.
Ran F.A., Cong L., Yan W.X., Scott D.A., Gootenberg J.S., Kriz A.J., Zetsche B., Shalem O., Wu X., Makarova K.S., Koonin E.V., Sharp P.A., Zhang F. (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature. 520, 186–191.
Kim E., Koo T., Park S.W., Kim D., Kim K., Cho H.-Y., Song D.W., Lee K.J., Jung M.H., Kim S., Kim J.H., Kim J.H., Kim J.-S. (2017) In vivo genome editing with a small Cas9 orthologue derived from Campylobacter j-ejuni. Nat. Commun. 8, 14500.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685.
Selkova P., Vasileva A., Pobegalov G., Musharova O., Arseniev A., Kazalov M., Zyubko T., Shcheglova N., Artamonova T., Khodorkovskii M., Severinov K., Fedorova I. (2020) Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA. RNA Biol. 17, 1472–1479.
Sun P., Tropea J.E., Waugh D.S. (2011) Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner. Methods Mol. Biol. 705, 259–274.
Esvelt K.M., Mali P., Braff J.L., Moosburner M., Yaung S.J., Church G.M. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods. 10, 1116–1121.
Zetsche B., Gootenberg J.S., Abudayyeh O.O., Slaymaker I.M., Makarova K.S., Essletzbichler P., Volz S.E., Joung J., van der Oost J., Regev A., Koonin E.V., Zhang F. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163, 759–771.
Crooks G.E., Hon G., Chandonia J.-M., Brenner S.E. (2004) WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190.
Hu Z., Wang S., Zhang C., Gao N., Li M., Wang D., Wang D., Liu D., Liu H., Ong S.-G., Wang H., Wang Y. (2020) A compact Cas9 ortholog from Staphylococcus auricularis (SauriCas9) expands the DNA targeting scope. PLoS Biol. 18, e3000686.
Leenay R.T., Maksimchuk K.R., Slotkowski R.A., Agrawal R.N., Gomaa A.A., Briner A.E., Barrangou R., Beisel C.L. (2016) Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell. 62, 137–47.
Cui Z., Tian R., Huang Z., Jin Z., Li L., Liu J., Huang Z., Xie H., Liu D., Mo H., Zhou R., Lang B., Meng B., Weng H., Hu Z. (2022) FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity. Nat. Commun. 13, 1425.
Fedorova I., Arseniev A., Selkova P., Pobegalov G., Goryanin I., Vasileva A., Musharova O., Abramova M., Kazalov M., Zyubko T., Artamonova T., Artamonova D., Shmakov S., Khodorkovskii M., Severinov K. (2020) DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 type II-C system. Nucleic Acids Res. 48, 2026–2034.