Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
23 06 2022
23 06 2022
Historique:
received:
19
07
2021
accepted:
16
06
2022
entrez:
23
6
2022
pubmed:
24
6
2022
medline:
28
6
2022
Statut:
epublish
Résumé
The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.
Identifiants
pubmed: 35739111
doi: 10.1038/s41467-022-31386-1
pii: 10.1038/s41467-022-31386-1
pmc: PMC9226073
doi:
Substances chimiques
RNA, Guide
0
Exonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3604Informations de copyright
© 2022. The Author(s).
Références
Jinek, M. et al. A programmable dual-RNA – guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–822 (2012).
pubmed: 22745249
pmcid: 6286148
doi: 10.1126/science.1225829
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
pubmed: 24906146
pmcid: 4343198
doi: 10.1016/j.cell.2014.05.010
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
pubmed: 23287722
pmcid: 3712628
doi: 10.1126/science.1232033
Pickar-oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).
pubmed: 31147612
pmcid: 7079207
doi: 10.1038/s41580-019-0131-5
Doudna, J. & Knott, G. CRISPR-Cas guides the future of genetic engineering. Science 361, 1–4 (2018).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
pubmed: 24529477
pmcid: 4139937
doi: 10.1016/j.cell.2014.02.001
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 1–18 (2016).
doi: 10.1038/nature17664
Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2017).
doi: 10.1038/ncb3452
Wang, H., La Russa, M. & Qi, L. S. CRISPR/Cas9 in genome editing and beyond. Annu. Rev. Biochem. 85, 227–264 (2016).
pubmed: 27145843
doi: 10.1146/annurev-biochem-060815-014607
Xie, S., Shen, B., Zhang, C., Huang, X. & Zhang, Y. SgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS ONE 9, 1–9 (2014).
Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).
pubmed: 25184501
pmcid: 4262738
doi: 10.1038/nbt.3026
Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).
pubmed: 24770324
pmcid: 4263420
doi: 10.1038/nbt.2909
Ran, F. A. et al. Double nicking by RNA-guided CRISPR cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).
pubmed: 23992846
pmcid: 3856256
doi: 10.1016/j.cell.2013.08.021
Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
pubmed: 26735016
pmcid: 4851738
doi: 10.1038/nature16526
Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).
pubmed: 29431739
pmcid: 6066108
doi: 10.1038/nbt.4066
Morisaka, H. et al. CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat. Commun. 10, 1–13 (2019).
doi: 10.1038/s41467-019-13226-x
Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).
pubmed: 28976959
pmcid: 5706658
doi: 10.1038/nature24049
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
pubmed: 26422227
pmcid: 4638220
doi: 10.1016/j.cell.2015.09.038
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
pubmed: 31634902
pmcid: 6907074
doi: 10.1038/s41586-019-1711-4
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
pubmed: 27096365
pmcid: 4873371
doi: 10.1038/nature17946
Tran, N. T. et al. Enhancement of precise gene editing by the association of Cas9 with homologous recombination factors. Front. Genet. 10, 1–13 (2019).
doi: 10.3389/fgene.2019.00365
Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).
pubmed: 25643054
doi: 10.1038/nbt.3149
Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).
pubmed: 25848930
pmcid: 4402137
doi: 10.1038/nchembio.1793
Shevelev, I. V. & Hübscher, U. The 3′−5′ exonucleases. Nat. Rev. Mol. Cell Biol. 3, 364–375 (2002).
pubmed: 11988770
doi: 10.1038/nrm804
Lovett, S. T. The DNA exonucleases of Escherichia coli. EcoSal Plus 4, 1–30 (2011).
doi: 10.1128/ecosalplus.4.4.7
Delacôte, F. et al. High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes. PLoS ONE 8, 1–8 (2013).
doi: 10.1371/journal.pone.0053217
Certo, M. T. et al. Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat. Methods 9, 973–975 (2012).
pubmed: 22941364
pmcid: 3602999
doi: 10.1038/nmeth.2177
Mashimo, T. et al. Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci. Rep. 3, 1–6 (2013).
doi: 10.1038/srep01253
Clements, T. P., Tandon, B., Lintel, H. A., McCarty, J. H. & Wagner, D. S. RICE CRISPR: Rapidly increased cut ends by an exonuclease Cas9 fusion in zebrafish. Genesis 55, 1–6 (2017).
doi: 10.1002/dvg.23044
Zhang, Q. et al. Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites. Sci. China Life Sci. https://doi.org/10.1007/s11427-020-1671-6 (2020).
doi: 10.1007/s11427-020-1671-6
pubmed: 33355886
pmcid: 7756132
Burkhard, P., Stetefeld, J. & Strelkov, S. V. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82–88 (2001).
pubmed: 11166216
doi: 10.1016/S0962-8924(00)01898-5
Ljubetič, A. et al. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 35, 1094–1101 (2017).
pubmed: 29035374
doi: 10.1038/nbt.3994
Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362–366 (2013).
pubmed: 23624438
pmcid: 3661711
doi: 10.1038/nchembio.1248
Thomson, A. R. et al. Computational design of water-soluble α-helical barrels. Science 280, 485–488 (2014).
doi: 10.1126/science.1257452
Fink, T. et al. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat. Chem. Biol. 15, 115–122 (2019).
pubmed: 30531965
doi: 10.1038/s41589-018-0181-6
Lebar, T., Lainšček, D., Merljak, E., Aupič, J. & Jerala, R. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat. Chem. Biol. 16, 513–519 (2020).
pubmed: 31907374
pmcid: 7182445
doi: 10.1038/s41589-019-0443-y
Cox, D. B. T., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
pubmed: 25654603
pmcid: 4492683
doi: 10.1038/nm.3793
Lainšček, D. et al. Delivery of an artificial transcription regulator dCas9-VPR by extracellular vesicles for therapeutic gene activation. ACS Synth. Biol. 7, 2715–2725 (2018).
pubmed: 30513193
doi: 10.1021/acssynbio.8b00192
Bjornsti, M. A. & Megonigal, M. D. Resolution of DNA molecules by one-dimensional agarose-gel electrophoresis. In Methods in molecular biology (Clifton, N.J.) 94, (Humana Press, 1999).
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).
pubmed: 24476820
pmcid: 4106473
doi: 10.1038/nature13011
Raper, A. T., Stephenson, A. A. & Suo, Z. Functional insights revealed by the kinetic mechanism of CRISPR/Cas9. J. Am. Chem. Soc. 140, 2971–2984 (2018).
pubmed: 29442507
doi: 10.1021/jacs.7b13047
Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature https://doi.org/10.1038/nature13589 (2014).
doi: 10.1038/nature13589
pubmed: 25470054
pmcid: 4297536
Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
pubmed: 25263330
pmcid: 4265475
doi: 10.1016/j.cell.2014.09.014
Tjaša Plaper, et al. Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike 1 protein-mediated cell fusion. Sci. Rep. 11, 1–23 (2020).
Drobnak, I., Gradišar, H., Ljubetič, A., Merljak, E. & Jerala, R. Modulation of coiled-coil dimer stability through surface residues while preserving pairing specificity. J. Am. Chem. Soc. 139, 8229–8236 (2017).
pubmed: 28553984
doi: 10.1021/jacs.7b01690
Plaper, T. et al. Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion. Sci. Rep. 11, 1–16 (2021).
doi: 10.1038/s41598-021-88315-3
Liu, Z. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep. 7, 1–9 (2017).
Kochanek, S., Schiedner, G. & Volpers, C. High-capacity ‘gutless’ adenoviral vectors. Curr. Opin. Mol. Ther. 3, 454–463 (2001).
pubmed: 11699889
Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets HHS public access. Nat. Methods 14, 607–614 (2017).
pubmed: 28459458
pmcid: 5924695
doi: 10.1038/nmeth.4278
Lazzarotto, C. R. et al. Defining CRISPR– Cas9 genome-wide nuclease activities with CIRCLE-seq. Nat. Protoc. 13, 2615–2642 (2018).
pubmed: 30341435
pmcid: 6512799
doi: 10.1038/s41596-018-0055-0
Pickar-Oliver, A. et al. Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy. Mol. Ther. 29, 3243–3257 (2021).
pubmed: 34509668
doi: 10.1016/j.ymthe.2021.09.003
Lazzarotto, C. R. et al. CHANGE-seq reveals genetic and epigenetic effects on CRISPR-Cas9 genome-wide activity. Nat. Biotechnol. 38, 1317–1327 (2020).
pubmed: 32541958
pmcid: 7652380
doi: 10.1038/s41587-020-0555-7
Maeder, M. L. & Gersbach, C. A. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430–446 (2016).
pubmed: 26755333
pmcid: 4786923
doi: 10.1038/mt.2016.10
Chen, Y. & Zhang, Y. Application of the CRISPR/Cas9 system to drug resistance in breast cancer. Adv. Sci. 5, 1–13 (2018).
doi: 10.1002/advs.201700964
Reckel, S. & Hantschel, O. Bcr-Abl: one kinase, two isoforms, two diseases. Oncotarget 8, 78257–78258 (2017).
pubmed: 29108223
pmcid: 5667957
doi: 10.18632/oncotarget.20874
García-Tuñón, I. et al. The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia. Oncotarget 8, 26027–26040 (2017).
pubmed: 28212528
pmcid: 5432235
doi: 10.18632/oncotarget.15215
Jiao, Q. et al. Advances in studies of tyrosine kinase inhibitors and their acquired resistance. Mol. Cancer 17, 1–12 (2018).
doi: 10.1186/s12943-018-0801-5
Su, S. et al. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci. Rep. 6, 1–14 (2016).
Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 367, 1–20 (2020).
doi: 10.1126/science.aba7365
Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).
pubmed: 25119044
pmcid: 4199937
doi: 10.1038/nature13589
Jabbour, E. ANNUAL CLINICAL UPDATES IN HEMATOLOGICAL Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring. 442–459 (2018). https://doi.org/10.1002/ajh.25011
Fine, E. J. et al. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci. Rep. 5, 1–9 (2015).
doi: 10.1038/srep10777
Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015).
pubmed: 25713377
pmcid: 4364227
doi: 10.1073/pnas.1501698112
Schmidt, R. et al. CRISPR activation and interference screens decode stimulation responses in primary human T cells. Science 375, 1–14 (2022).
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 1–13 (2021).
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97 (2020).
pubmed: 31937940
pmcid: 6980783
doi: 10.1038/s41551-019-0501-5
Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).
pubmed: 27815355
doi: 10.1158/1078-0432.CCR-16-1300
Seki, A. & Rutz, S. Optimized RNP transfection for highly efficient CRISPR / Cas9-mediated gene knockout in primary T cells. J. Exp. Med. 1–13 (2018). https://doi.org/10.1084/jem.20171626
Lu, Y. et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat. Med. 26, 732–740 (2020).
pubmed: 32341578
doi: 10.1038/s41591-020-0840-5
Brinkman, E. K., Chen, T., Amendola, M. & Van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, 1–8 (2014).
doi: 10.1093/nar/gku936
Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 34, 695–697 (2016).
pubmed: 27404874
pmcid: 5242601
doi: 10.1038/nbt.3583
Clement, K. et al. Accurate and rapid analysis of genome editing data from nucleases and base editors with CRISPResso2. Nat. Biotechnol. 37, 224 (2019).
pubmed: 30809026
pmcid: 6533916
doi: 10.1038/s41587-019-0032-3