Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening

Michael Aregger, Thomas Gonatopoulos-Pournatzis, Kun Xing

Published: 2021-09-09 DOI: 10.1038/s41596-021-00595-1

CRISPR

genetic screening

CHyMErA

Cas9-Cas12a

combinatorial genome-editing

AI 解读

Fabrication of angstrom-scale two-dimensional channels for mass transport

Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells

The eINTACT method for studying nuclear changes in host plant cells targeted by bacterial effectors in native infection contexts

Rapid reaction optimization by robust and economical quantitative benchtop19F NMR spectroscopy

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures

Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds

Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes

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CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic per
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
ACKNOWLEDGMENTS

Knott, G. J. & Doudna, J. A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
Hille, F. et al. The biology of CRISPR-Cas: backward and forward. Cell 172, 1239–1259 (2018).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Haley, B. & Roudnicky, F. Functional genomics for cancer drug target discovery. Cancer Cell 38, 31–43 (2020).
Doench, J. G. Am i ready for CRISPR? A user’s guide to genetic screens. Nat. Rev. Genet. 19, 67–80 (2018).
Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).
Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).
Ford, K., McDonald, D. & Mali, P. Functional genomics via CRISPR–Cas. J. Mol. Biol. 431, 48–65 (2019).
Costanzo, M. et al. Global genetic networks and the genotype-to-phenotype relationship. Cell 177, 85–100 (2019).
Gonatopoulos-Pournatzis, T. et al. Genome-wide CRISPR-Cas9 interrogation of splicing networks reveals a mechanism for recognition of autism-misregulated neuronal microexons. Mol. Cell 72, 510–524.e12 (2018).
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
Liu, Y. et al. CRISPR activation screens systematically identify factors that drive neuronal fate and reprogramming. Cell Stem Cell 23, 758–771.e8 (2018).
Patel, S. J. et al. Identification of essential genes for cancer immunotherapy. Nature 548, 537–542 (2017).
Park, R. J. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 49, 193–203 (2016).
Haney, M. S. et al. Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nat. Genet. 50, 1716–1727 (2018).
Lawson, K. A. et al. Functional genomic landscape of cancer-intrinsic evasion of killing by T cells. Nature 586, 120–126 (2020).
Tian, R. et al. CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons. Neuron 104, 239–255.e12 (2019).
Mair, B. et al. Essential gene profiles for human pluripotent stem cells identify uncharacterized genes and substrate dependencies. Cell Rep. 27, 599–615.e12 (2019).
Olivieri, M. et al. A genetic map of the response to DNA damage in human cells. Cell 182, 481–496.e21 (2020).
Horlbeck, M. A. et al. Mapping the genetic landscape of human cells. Cell 174, 953–967.e22 (2018).
Gonatopoulos-Pournatzis, T. et al. Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9–Cas12a platform. Nat. Biotechnol. 38, 638–648 (2020).
Najm, F. J. et al. Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens. Nat. Biotechnol. 36, 179–189 (2018).
Han, K. et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol. 35, 463–474 (2017).
Norman, T. M. et al. Exploring genetic interaction manifolds constructed from rich single-cell phenotypes. Science 365, 786–793 (2019).
DeWeirdt, P. C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat. Biotechnol. 39, 94–104 (2020).
Shen, J. P. et al. Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat. Methods 14, 573–576 (2017).
Irimia, M. et al. A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159, 1511–1523 (2014).
Kahles, A. et al. Comprehensive analysis of alternative splicing across tumors from 8,705 patients. Cancer Cell 34, 211–224.e6 (2018).
Gandal, M. J. et al. Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder. Science 362, eaat8127 (2018).
Calabrese, C. et al. Genomic basis for RNA alterations in cancer. Nature 578, 129–136 (2020).
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).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Zetsche, B. et al. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2016).
Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).
Konermann, S. et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell 173, 665–676.e14 (2018).
Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).
Wessels, H. H. et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol. 38, 722–727 (2020).
Thomas, J. D. et al. RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons. Nat. Genet. 52, 84–94 (2020).
Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016).
Zhao, D. et al. Combinatorial CRISPR-Cas9 metabolic screens reveal critical redox control points dependent on the KEAP1-NRF2 regulatory axis. Mol. Cell 69, 699–708.e7 (2018).
Boettcher, M. et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. Nat. Biotechnol. 36, 170–178 (2018).
Liu, J. et al. Pooled library screening with multiplexed Cpf1 library. Nat. Commun. 10, 3144 (2019).
Dede, M., McLaughlin, M., Kim, E. & Hart, T. Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens. Genome Biol. 21, 262 (2020).
Chow, R. D. et al. In vivo profiling of metastatic double knockouts through CRISPR–Cpf1 screens. Nat. Methods 16, 405–408 (2019).
Gier, R. A. et al. High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening. Nat. Commun. 11, 3455 (2020).
ter Brake, O. et al. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol. Ther. 16, 557–564 (2008).
Sack, L. M., Davoli, T., Xu, Q., Li, M. Z. & Elledge, S. J. Sources of error in mammalian genetic screens. G3 (Bethesda) 6, 2781–2790 (2016).
Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016).
Vidigal, J. A. & Ventura, A. Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nat. Commun. 6, 8083 (2015).
Brown, K. R., Mair, B., Soste, M. & Moffat, J. CRISPR screens are feasible in TP53 wild‐type cells. Mol. Syst. Biol. 15, e8679 (2019).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Ihry, R. J. et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).
Bowden, A. R. et al. Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance. Elife 9, e55325 (2020).
Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife 5, e19760 (2016).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442 (2013).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Tak, Y. E. et al. Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors. Nat. Methods 14, 1163–1166 (2017).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Hanna, R. E. & Doench, J. G. A case of mistaken identity. Nat. Biotechnol. 36, 802–804 (2018).
Feldman, D., Singh, A., Garrity, A. J. & Blainey, P. C. Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens. Preprint at bioRxiv https://doi.org/10.1101/262121 (2018).
Hegde, M., Strand, C., Hanna, R. E. & Doench, J. G. Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens. PLoS One 13, e0197547 (2018).
Hart, T. et al. Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens. G3 (Bethesda) 7, 2719–2727 (2017).
Ward, H. N. et al. Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline. Nat. Protoc. https://doi.org/10.1038/s41596-021-00596-0 (2021).
Aregger, M. et al. Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism. Nat. Metab. 2, 499–513 (2020).
Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017).
Behan, F. M. et al. Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568, 511–516 (2019).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).
Jost, M. et al. Combined CRISPRi/a-based chemical genetic screens reveal that rigosertib is a microtubule-destabilizing agent. Mol. Cell 68, 210–223.e6 (2017).
Mair, B. et al. High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting. Nat. Biomed. Eng. 3, 796–805 (2019).
Wroblewska, A. et al. Protein barcodes enable high-dimensional single-cell CRISPR screens. Cell 175, 1141–1155.e16 (2018).
Wang, B. et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat. Protoc. 14, 756–780 (2019).
Aregger, M., Chandrashekhar, M., Tong, A. H. Y., Chan, K. & Moffat, J. Pooled lentiviral CRISPR-Cas9 screens for functional genomics in mammalian cells. Methods Mol. Biol. 1869, 169–188 (2019).
de Kegel, B. & Ryan, C. J. Paralog buffering contributes to the variable essentiality of genes in cancer cell lines. PLoS Genet 15, e1008466 (2019).
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).
Huang, T. P., Newby, G. A. & Liu, D. R. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat. Protoc. 16, 1089–1128 (2021).
Sanson, K. R. et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416 (2018).
Listgarten, J. et al. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nat. Biomed. Eng. 2, 38–47 (2018).
Perez, A. R. et al. GuideScan software for improved single and paired CRISPR guide RNA design. Nat. Biotechnol. 35, 347–349 (2017).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Kim, H. K. et al. Deep learning improves prediction of CRISPR-Cpf1 guide RNA activity. Nat. Biotechnol. 36, 239–241 (2018).
Tycko, J. et al. Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nat. Commun. 10, 4063 (2019).
Perez, A. R., Sala, L., Perez, R. K. & Vidigal, J. A. Computational correction of off-targeting for CRISPR-Cas9 essentiality screens. Preprint at bioRxiv https://doi.org/10.1101/809970 (2020).

Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening

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