Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
23 Aug 2024
23 Aug 2024
Historique:
received:
24
01
2024
accepted:
15
08
2024
medline:
24
8
2024
pubmed:
24
8
2024
entrez:
23
8
2024
Statut:
epublish
Résumé
Type I CRISPR-Cas systems are widespread and have exhibited high versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems, with a total gene size smaller than that of SpCas9, can be developed for transcriptional activation in human cells. The efficiency of the engineered I-F2 tool can match or surpass that of dCas9. Additionally, we create a base editor using the I-F2 Cascade, which induces a considerably wide editing window (~30 nt) with a bimodal distribution. It can expand targetable sites, which is useful for disrupting functional sequences and genetic screening. This research underscores the application of compact type I systems in eukaryotes, particularly in the development of a base editor with a wide editing window.
Identifiants
pubmed: 39179566
doi: 10.1038/s41467-024-51695-x
pii: 10.1038/s41467-024-51695-x
doi:
Substances chimiques
CRISPR-Associated Protein 9
EC 3.1.-
RNA, Guide, CRISPR-Cas Systems
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7277Subventions
Organisme : National Natural Science Foundation of China (National Science Foundation of China)
ID : 32100499
Organisme : National Natural Science Foundation of China (National Science Foundation of China)
ID : 32150020
Organisme : National Natural Science Foundation of China (National Science Foundation of China)
ID : 32230061
Organisme : National Natural Science Foundation of China (National Science Foundation of China)
ID : 32150020
Informations de copyright
© 2024. The Author(s).
Références
Makarova, K. S. et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).
pubmed: 31857715
doi: 10.1038/s41579-019-0299-x
Hille, F. et al. The biology of CRISPR-Cas: backward and forward. Cell 172, 1239–1259 (2018).
pubmed: 29522745
doi: 10.1016/j.cell.2017.11.032
Xiao, Y. et al. Structure basis for directional R-loop formation and substrate handover mechanisms in Type I CRISPR-Cas system. Cell 170, 48–60.e11 (2017).
pubmed: 28666122
pmcid: 5841471
doi: 10.1016/j.cell.2017.06.012
Cheng, F. et al. Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon. J. Genet. Genomics 44, 541–548 (2017).
pubmed: 29169919
doi: 10.1016/j.jgg.2017.09.010
Luo, M. L., Mullis, A. S., Leenay, R. T. & Beisel, C. L. Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res. 43, 674–681 (2015).
pubmed: 25326321
doi: 10.1093/nar/gku971
Csorgo, B. et al. A compact Cascade-Cas3 system for targeted genome engineering. Nat. Methods 17, 1183–1190 (2020).
pubmed: 33077967
pmcid: 7611934
doi: 10.1038/s41592-020-00980-w
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).
pubmed: 31189177
doi: 10.1038/s41586-019-1323-z
Du, K., Gong, L., Li, M., Yu, H. & Xiang, H. Reprogramming the endogenous type I CRISPR‐Cas system for simultaneous gene regulation and editing in Haloarcula hispanica. mLife 1, 40–50 (2022).
pubmed: 38818324
pmcid: 10989794
doi: 10.1002/mlf2.12010
Cameron, P. et al. Harnessing type I CRISPR-Cas systems for genome engineering in human cells. Nat. Biotechnol. 37, 1471–1477 (2019).
pubmed: 31740839
doi: 10.1038/s41587-019-0310-0
Dolan, A. E. et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using Type I CRISPR-Cas. Mol. Cell 74, 936–950.e935 (2019).
pubmed: 30975459
pmcid: 6555677
doi: 10.1016/j.molcel.2019.03.014
Morisaka, H. et al. CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat. Commun. 10, 5302 (2019).
pubmed: 31811138
pmcid: 6897959
doi: 10.1038/s41467-019-13226-x
Hu, C. et al. Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools. Mol. Cell 82, 2754–2768.e2755 (2022).
pubmed: 35835111
pmcid: 9357151
doi: 10.1016/j.molcel.2022.06.007
Tan, R. et al. Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems. Mol. Cell 82, 852–867.e855 (2022).
pubmed: 35051351
pmcid: 8964063
doi: 10.1016/j.molcel.2021.12.032
Li, Y. et al. Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system. Plant Biotechnol. J. 21, 2196–2208 (2023).
pubmed: 37641539
pmcid: 10579709
doi: 10.1111/pbi.14122
Kita, Y. et al. Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs. Stem Cell Rep. 18, 1753–1765 (2023).
doi: 10.1016/j.stemcr.2023.07.007
Osakabe, K. et al. Genome editing in plants using CRISPR type I-D nuclease. Commun. Biol. 3, 648 (2020).
pubmed: 33159140
pmcid: 7648086
doi: 10.1038/s42003-020-01366-6
Osakabe, K., Wada, N., Murakami, E., Miyashita, N. & Osakabe, Y. Genome editing in mammalian cells using the CRISPR type I-D nuclease. Nucleic Acids Res. 49, 6347–6363 (2021).
pubmed: 34076237
pmcid: 8216271
doi: 10.1093/nar/gkab348
Zimmermann, A. et al. A Cas3-base editing tool for targetable in vivo mutagenesis. Nat. Commun. 14, 3389 (2023).
pubmed: 37296137
pmcid: 10256805
doi: 10.1038/s41467-023-39087-z
Chen, Y. et al. Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells. Nat. Commun. 11, 3136 (2020).
pubmed: 32561716
pmcid: 7305327
doi: 10.1038/s41467-020-16880-8
Young, J. K. et al. The repurposing of type I-E CRISPR-Cascade for gene activation in plants. Commun. Biol. 2, 383 (2019).
pubmed: 31646186
pmcid: 6802105
doi: 10.1038/s42003-019-0637-6
Pickar-Oliver, A. et al. Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells. Nat. Biotechnol. 37, 1493–1501 (2019).
pubmed: 31548729
pmcid: 6893126
doi: 10.1038/s41587-019-0235-7
Mendell, J. R. et al. Current clinical applications of in vivo gene therapy with AAVs. Mol. Ther. 29, 464–488 (2021).
pubmed: 33309881
doi: 10.1016/j.ymthe.2020.12.007
Szczelkun, M. D. et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl Acad. Sci. USA 111, 9798–9803 (2014).
pubmed: 24912165
pmcid: 4103346
doi: 10.1073/pnas.1402597111
Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).
pubmed: 26841432
pmcid: 5111852
doi: 10.1126/science.aad8282
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
Ye, L. et al. Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-02050-w (2024).
doi: 10.1038/s41587-023-02050-w
pubmed: 38918616
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
pubmed: 32572269
doi: 10.1038/s41587-020-0561-9
Han, D. et al. Development of miniature base editors using engineered IscB nickase. Nat. Methods 20, 1029–1036 (2023).
pubmed: 37231266
doi: 10.1038/s41592-023-01898-9
Swarts, D. C., van der Oost, J. & Jinek, M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a. Mol. Cell 66, 221–233.e224 (2017).
pubmed: 28431230
pmcid: 6879319
doi: 10.1016/j.molcel.2017.03.016
Pausch, P. et al. Structural variation of Type I-F CRISPR RNA guided DNA surveillance. Mol. Cell 67, 622–632.e624 (2017).
pubmed: 28781236
doi: 10.1016/j.molcel.2017.06.036
Dwarakanath, S. et al. Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens. Nucleic Acids Res 43, 8913–8923 (2015).
pubmed: 26350210
pmcid: 4605320
doi: 10.1093/nar/gkv882
Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).
pubmed: 21552286
doi: 10.1038/nrmicro2577
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
pubmed: 22745249
pmcid: 6286148
doi: 10.1126/science.1225829
McBride, T. M. et al. Diverse CRISPR-Cas complexes require independent translation of small and large subunits from a single gene. Mol. Cell 80, 971–979.e977 (2020).
pubmed: 33248026
doi: 10.1016/j.molcel.2020.11.003
Klompe, S. E. et al. Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons. Mol. Cell 82, 616–628.e615 (2022).
pubmed: 35051352
pmcid: 8849592
doi: 10.1016/j.molcel.2021.12.021
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
pubmed: 25730490
pmcid: 4393883
doi: 10.1038/nmeth.3312
Kuznedelov, K. et al. Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. Nucleic Acids Res. 44, 10849–10861 (2016).
pubmed: 27738137
pmcid: 5159557
doi: 10.1093/nar/gkw914
Gleditzsch, D. et al. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system. Nucleic Acids Res. 44, 5872–5882 (2016).
pubmed: 27216815
pmcid: 4937334
doi: 10.1093/nar/gkw469
Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).
pubmed: 32433547
pmcid: 7357821
doi: 10.1038/s41587-020-0453-z
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
pubmed: 29160308
pmcid: 5726555
doi: 10.1038/nature24644
Lettre, G. & Bauer, D. E. Fetal haemoglobin in sickle-cell disease: from genetic epidemiology to new therapeutic strategies. Lancet 387, 2554–2564 (2016).
pubmed: 27353686
doi: 10.1016/S0140-6736(15)01341-0
Sankaran, V. G. & Orkin, S. H. The switch from fetal to adult hemoglobin. Cold Spring Harb. Perspect. Med. 3, a011643 (2013).
pubmed: 23209159
pmcid: 3530042
doi: 10.1101/cshperspect.a011643
Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).
pubmed: 26375006
pmcid: 4644101
doi: 10.1038/nature15521
Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).
pubmed: 30911135
pmcid: 6512986
doi: 10.1038/s41591-019-0401-y
Fu, B. et al. CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric beta
pubmed: 35922667
doi: 10.1038/s41591-022-01906-z
Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).
pubmed: 28585549
pmcid: 5467206
doi: 10.1038/ncomms15790
Davis, J. R. et al. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors. Nat. Biomed. Eng. 6, 1272–1283 (2022).
pubmed: 35902773
pmcid: 9652153
doi: 10.1038/s41551-022-00911-4
Altae-Tran, H. et al. Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science 382, eadi1910 (2023).
pubmed: 37995242
pmcid: 10910872
doi: 10.1126/science.adi1910
Gong, L. et al. Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica. Nucleic Acids Res. 47, 5880–5891 (2019).
pubmed: 30957847
pmcid: 6582329
doi: 10.1093/nar/gkz244
Xu, X. et al. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol. Cell 81, 4333–4345.e4334 (2021).
pubmed: 34480847
doi: 10.1016/j.molcel.2021.08.008
Li, J. et al. Plant base editing and prime editing: The current status and future perspectives. J. Integr. Plant Biol. 65, 444–467 (2023).
pubmed: 36479615
doi: 10.1111/jipb.13425
Chen, W. et al. Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors. Mol. Cell 83, 2768–2780.e2766 (2023).
pubmed: 37402371
doi: 10.1016/j.molcel.2023.06.014
Caldecott, K. W. Causes and consequences of DNA single-strand breaks. Trends Biochem. Sci. 49, 68–78 (2024).
pubmed: 38040599
doi: 10.1016/j.tibs.2023.11.001
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
pubmed: 25513782
doi: 10.1038/nbt.3117
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).
pubmed: 24463574
pmcid: 3988262
doi: 10.1038/nbt.2808
Chen, L. et al. Short- and long-read metagenomics expand individualized structural variations in gut microbiomes. Nat. Commun. 13, 3175 (2022).
pubmed: 35676264
pmcid: 9177567
doi: 10.1038/s41467-022-30857-9
Almeida, A. et al. A new genomic blueprint of the human gut microbiota. Nature 568, 499–504 (2019).
pubmed: 30745586
pmcid: 6784870
doi: 10.1038/s41586-019-0965-1
Zou, Y. et al. 1520 reference genomes from cultivated human gut bacteria enable functional microbiome analyses. Nat. Biotechnol. 37, 179–185 (2019).
pubmed: 30718868
pmcid: 6784896
doi: 10.1038/s41587-018-0008-8
Minh, B. Q. et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).
pubmed: 32011700
pmcid: 7182206
doi: 10.1093/molbev/msaa015
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
doi: 10.14806/ej.17.1.200
Varadi, M. et al. AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022).
pubmed: 34791371
doi: 10.1093/nar/gkab1061
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
pubmed: 34265844
pmcid: 8371605
doi: 10.1038/s41586-021-03819-2
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
pubmed: 24463181
pmcid: 4016707
doi: 10.1093/bioinformatics/btu048
Wilm, A. et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 40, 11189–11201 (2012).
pubmed: 23066108
pmcid: 3526318
doi: 10.1093/nar/gks918
Chen, T. et al. The genome sequence archive family: toward explosive data growth and diverse data types. Genom. Proteom. Bioinforma. 19, 578–583 (2021).
doi: 10.1016/j.gpb.2021.08.001
CNCB-NGDC members and partners. Database resources of the National Genomics Data Center, China National Center for Bioinformation in 2022. Nucleic Acids Res. 50, D27–D38 (2022).
Yu, H. Calculate the coverage of sRNA-seq reads from BLAST output and used to split fastq barcode samples. Zenodo. https://doi.org/10.5281/zenodo.12748513 (2024).