Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
09 2019
09 2019
Historique:
received:
23
01
2019
accepted:
07
06
2019
pubmed:
14
8
2019
medline:
9
11
2019
entrez:
14
8
2019
Statut:
ppublish
Résumé
The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.
Identifiants
pubmed: 31406383
doi: 10.1038/s41592-019-0508-6
pii: 10.1038/s41592-019-0508-6
doi:
Substances chimiques
RNA, Guide
0
Endonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
887-893Subventions
Organisme : Brain and Behavior Research Foundation (Brain & Behavior Research Foundation)
ID : 26606
Pays : International
Références
Knott, G. J. & Doudna, J. A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
doi: 10.1126/science.aat5011
Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).
doi: 10.1038/nbt.2842
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
doi: 10.1038/nature14136
Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).
doi: 10.1038/nrg3899
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
doi: 10.1016/j.cell.2013.06.044
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
doi: 10.1038/nbt.2675
Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
doi: 10.1038/nmeth.2598
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
doi: 10.1038/nmeth.2600
Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).
doi: 10.1038/cr.2013.122
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
doi: 10.1016/j.cell.2013.02.022
Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).
doi: 10.1016/j.cell.2014.11.052
Boettcher, M. et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. Nat. Biotechnol. 36, 170–178 (2018).
doi: 10.1038/nbt.4062
Dahlman, J. E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).
doi: 10.1038/nbt.3390
Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).
doi: 10.1038/nmeth.3580
Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).
doi: 10.1093/nar/gkt520
Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).
doi: 10.1016/j.molcel.2016.02.031
Rogers, J. K. & Church, G. M. Multiplexed engineering in biology. Trends Biotechnol. 34, 198–206 (2016).
doi: 10.1016/j.tibtech.2015.12.004
Xie, K., Minkenberg, B. & Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).
doi: 10.1073/pnas.1420294112
Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. & Lu, T. K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).
doi: 10.1016/j.molcel.2014.04.022
Ruiz-Mirazo, K., Briones, C. & de la Escosura, A. Chemical roots of biological evolution: the origins of life as a process of development of autonomous functional systems. Open Biol. 7, 170050 (2017).
doi: 10.1098/rsob.170050
Fonfara, I., Richter, H., Bratovic, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).
doi: 10.1038/nature17945
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
doi: 10.1016/j.cell.2015.09.038
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).
doi: 10.1016/j.molcel.2017.03.016
Wu, W. Y., Lebbink, J. H. G., Kanaar, R., Geijsen, N. & van der Oost, J. Genome editing by natural and engineered CRISPR-associated nucleases. Nat. Chem. Biol. 14, 642–651 (2018).
doi: 10.1038/s41589-018-0080-x
Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).
doi: 10.1038/nbt.3737
Zhong, G., Wang, H., Li, Y., Tran, M. H. & Farzan, M. Cpf1 proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells. Nat. Chem. Biol. 13, 839–841 (2017).
doi: 10.1038/nchembio.2410
Tak, Y. E. et al. Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors. Nat. Methods 14, 1163–1166 (2017).
doi: 10.1038/nmeth.4483
Arimbasseri, A. G., Rijal, K. & Maraia, R. J. Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation. Transcription 5, e27639 (2014).
doi: 10.4161/trns.27369
Wilusz, J. E. et al. A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. Genes Dev. 26, 2392–2407 (2012).
doi: 10.1101/gad.204438.112
Meyers, R. A. Encyclopedia of Molecular Cell Biology and Molecular Medicine 2nd edn (Wiley-VCH, 2004).
Huntley, S. et al. A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors. Genome Res. 16, 669–677 (2006).
doi: 10.1101/gr.4842106
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
doi: 10.1038/nmeth.3312
Singh, D. et al. Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc. Natl Acad. Sci. USA 115, 5444–5449 (2018).
doi: 10.1073/pnas.1718686115
Schmidt, F. & Platt, R. J. Applications of CRISPR-Cas for synthetic biology and genetic recording. Curr. Opin. Syst. Biol. 5, 9–15 (2017).
doi: 10.1016/j.coisb.2017.05.008
Sheth, R. U. & Wang, H. H. DNA-based memory devices for recording cellular events. Nat. Rev. Genet. 19, 718–732 (2018).
doi: 10.1038/s41576-018-0052-8
Farzadfard, F. & Lu, T. K. Emerging applications for DNA writers and molecular recorders. Sci. 361, 870–875 (2018).
doi: 10.1126/science.aat9249
Tennyson, C. N., Klamut, H. J. & Worton, R. G. The human dystrophin gene requires 16 h to be transcribed and is cotranscriptionally spliced. Nat. Genet. 9, 184–190 (1995).
doi: 10.1038/ng0295-184
Zoephel, J. & Randau, L. RNA-Seq analyses reveal CRISPR RNA processing and regulation patterns. Biochem. Soc. Trans. 41, 1459–1463 (2013).
doi: 10.1042/BST20130129
Liao, C., Slotkowski, R. A., Achmedov, T. & Beisel, C. L. The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays. RNA Biol. 16, 1–9 (2018).
doi: 10.1080/15476286.2018.1557498
Liao, C. et al. One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/312421v1 (2018).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008).
doi: 10.1038/nprot.2008.73
Heidrich, N., Dugar, G., Vogel, J., Sharma, C. M. & Investigating CRISPR, R. N. A. Biogenesis and function using RNA-seq. Methods Mol. Biol. 1311, 1–21 (2015).
doi: 10.1007/978-1-4939-2687-9_1
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10 (2011).
doi: 10.14806/ej.17.1.200
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
doi: 10.1186/gb-2009-10-3-r25
Quinlan, A. R. BEDTools: the Swiss-army tool for genome feature analysis. Curr. Protoc. Bioinforma. 47, 11.12.11–11.12.34 (2014).
doi: 10.1002/0471250953.bi1112s47
Roa, W. et al. Identification of a new microRNA expression profile as a potential cancer screening tool. Clin. Invest. Med. 33, E124 (2010).
doi: 10.25011/cim.v33i2.12351
Hui, A. B. et al. Comprehensive microRNA profiling for head and neck squamous cell carcinomas. Clin. Cancer Res. 16, 1129–1139 (2010).
doi: 10.1158/1078-0432.CCR-09-2166
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
doi: 10.1038/s41587-019-0032-3