Abstract
CRISPR activation provides an invaluable tool for experimental biologists to convert correlations into causation by directly observing phenotypic changes upon targeted changes in gene expression. With few exceptions, most diseases are caused by complex polygenic interactions, with multiple genes contributing to define the output of a gene network. As such researchers are increasingly interested in tools that can offer not only control but also the capacity to simultaneously upregulate multiple genes. The adaptation of CRISPR/Cas12a has provided a system especially suited to the tightly coordinated overexpression of multiple targeted genes. Here we describe an approach to test for active targeting crRNAs for dFnCas12a-VPR, before proceeding to generate and validate longer crRNA arrays for multiplexed targeting of genes of interest.
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References
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Gilbert LA, Larson MH, Morsut L et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–451
Chakraborty S, Ji H, Kabadi AM et al (2014) A CRISPR/Cas9-based system for reprogramming cell lineage specification. Stem Cell Rep 3:940–947
Nakamura M, Srinivasan P, Chavez M et al (2019) Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells. Nat Commun 10:194
Krawczyk K, Scheller L, Kim H et al (2020) Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming. Nat Commun 11:608
Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771
Campa CC, Weisbach NR, Santinha AJ et al (2019) Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods 16:887–893
Bryson JW, Auxillos JY, Rosser SJ (2021) Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor. Nucleic Acids Res 50:549–560
Tu M, Lin L, Cheng Y et al (2017) A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res 45:11295–11304
Tak YE, Kleinstiver BP, Nuñez JK et al (2017) Inducible and multiplex gene regulation using CRISPR–Cpf1-based transcription factors. Nat Methods 14:1163–1166
Kim HK, Min S, Song M et al (2018) Deep learning improves prediction of CRISPR–Cpf1 guide RNA activity. Nat Biotechnol 36:239–241
Martin FJ, Amode MR, Aneja A et al (2022) Ensembl 2023. Nucleic Acids Res gkac958
Kent WJ, Sugnet CW, Furey TS et al (2002) The human genome browser at UCSC. Genome Res 12:996–1006
Marshall R, Maxwell CS, Collins SP et al (2018) Rapid and scalable characterization of CRISPR technologies using an E. Coli cell-free transcription-translation. System 69:146
Reyes A, Huber W (2018) Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues. Nucleic Acids Res 46:582–592
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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Bryson, J.W., Rosser, S.J. (2024). Multiplexed Transactivation of Mammalian Cells Using dFnCas12a-VPR. In: Ceroni, F., Polizzi, K. (eds) Mammalian Synthetic Systems. Methods in Molecular Biology, vol 2774. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3718-0_13
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DOI: https://doi.org/10.1007/978-1-0716-3718-0_13
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