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Review: A systematic guide for identifying transcription factors that directly regulate the expression of a gene of interest
- Andrew D. Bates,
- Dawid Grzela,
- Maciej Studzian,
- Louise Brennan,
- Moli Williams,
- Conor Fawcett,
- Beth Hammond,
- Manreen Grewal,
- Marcin Ratajewski,
- Lukasz Pulaski,
- and Urszula L. McClurg
Genome Res. March 2026 36: 433-459; Published in Advance February 17, 2026, doi:10.1101/gr.281154.125
...in incomplete knockdown and can suffer from off-target effects, which complicate interpretation of transcriptional changes.Genome editing approaches, such as CRISPR–Cas9-mediated knockout (Nakamura et al. 2021), enable permanent removal of a TF. Knockouts are powerful for defining essential regulatory roles...
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Method: Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts
- Irmgard U. Haussmann,
- Thomas C. Dix,
- David W.J. McQuarrie,
- Veronica Dezi,
- Abdullah I. Hans,
- Roland Arnold,
- and Matthias Soller
Genome Res. December 2024 34: 2279-2292; Published in Advance December 3, 2024, doi:10.1101/gr.279479.124
...RNA structure would impact on Cas9 cleavage efficiency has not systematically been analyzed (Riesenberg et al. 2023). In particular, highly GC-rich gRNAs could disrupt the rather weak secondary structure of the sgRNA bound by Cas9.The CRISPR–Cas9 editing tool is widely used to generate knockout mutants...
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Method: High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori
- Sanyuan Ma,
- Tong Zhang,
- Ruolin Wang,
- Pan Wang,
- Yue Liu,
- Jiasong Chang,
- Aoming Wang,
- Xinhui Lan,
- Le Sun,
- Hao Sun,
- Run Shi,
- Wei Lu,
- Dan Liu,
- Na Zhang,
- Wenbo Hu,
- Xiaogang Wang,
- Weiqing Xing,
- Ling Jia,
- and Qingyou Xia
Genome Res. January 2024 34: 134-144; Published in Advance January 8, 2024, doi:10.1101/gr.278297.123
...screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori. Genome Res 30: 757–767. doi:10.1101/gr.249045.119 ↵Chang J, Chen X, Zhang T, Wang R, Wang A, Lan X, Zhou Y, Ma S, Xia Q. 2020b. The novel insight into the outcomes of CRISPR/Cas9 editing intra...
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Method: Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing
- Kepin Wang,
- Qin Jin,
- Degong Ruan,
- Yi Yang,
- Qishuai Liu,
- Han Wu,
- Zhiwei Zhou,
- Zhen Ouyang,
- Zhaoming Liu,
- Yu Zhao,
- Bentian Zhao,
- Quanjun Zhang,
- Jiangyun Peng,
- Chengdan Lai,
- Nana Fan,
- Yanhui Liang,
- Ting Lan,
- Nan Li,
- Xiaoshan Wang,
- Xinlu Wang,
- Yong Fan,
- Pieter A. Doevendans,
- Joost P.G. Sluijter,
- Pentao Liu,
- Xiaoping Li,
- and Liangxue Lai
Genome Res. December 2017 27: 2061-2071; Published in Advance November 16, 2017, doi:10.1101/gr.222521.117
...can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of -edited pigs for disease modeling.The CRISPR-Cas9 system is a powerful editing technology that uses the endonuclease Cas9 and single-guide RNAs (sg...
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Method: High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
- Gaurav K. Varshney,
- Wuhong Pei,
- Matthew C. LaFave,
- Jennifer Idol,
- Lisha Xu,
- Viviana Gallardo,
- Blake Carrington,
- Kevin Bishop,
- MaryPat Jones,
- Mingyu Li,
- Ursula Harper,
- Sunny C. Huang,
- Anupam Prakash,
- Wenbiao Chen,
- Raman Sood,
- Johan Ledin,
- and Shawn M. Burgess
Genome Res. July 2015 25: 1030-1042; Published in Advance June 5, 2015, doi:10.1101/gr.186379.114
...-throughput mutagenesis projects because of the simple design requirements, ease of use, and the ability to simultaneously target multiple genes. While the application of CRISPR/Cas9 in zebrafish editing has been shown (Chang et al. 2013; Hwang et al. 2013a,b; Jao et al. 2013; Gagnon et al. 2014); high-throughput methods...
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Method: CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins
- Daniel Savic,
- E. Christopher Partridge,
- Kimberly M. Newberry,
- Sophia B. Smith,
- Sarah K. Meadows,
- Brian S. Roberts,
- Mark Mackiewicz,
- Eric M. Mendenhall,
- and Richard M. Myers
Genome Res. October 2015 25: 1581-1589; Published in Advance September 9, 2015, doi:10.1101/gr.193540.115
...epeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR e pitope t agging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma...
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Method: Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations
- Jonathan S. Brammeld,
- Mia Petljak,
- Inigo Martincorena,
- Steven P. Williams,
- Luz Garcia Alonso,
- Alba Dalmases,
- Beatriz Bellosillo,
- Carla Daniela Robles-Espinoza,
- Stacey Price,
- Syd Barthorpe,
- Patrick Tarpey,
- Constantine Alifrangis,
- Graham Bignell,
- Joana Vidal,
- Jamie Young,
- Lucy Stebbings,
- Kathryn Beal,
- Michael R. Stratton,
- Julio Saez-Rodriguez,
- Mathew Garnett,
- Clara Montagut,
- Francesco Iorio,
- and Ultan McDermott
Genome Res. April 2017 27: 613-625; Published in Advance February 8, 2017, doi:10.1101/gr.213546.116
.... Such screens, if sufficiently unbiased, could in theory capture the entire breadth of genetic resistance mechanisms for any drug. Recent studies have demonstrated the power of both -wide gain- and loss-offunction screens using CRISPR/Cas9, lentiviral shRNA, and largescale open-reading frame technologies...
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Perspective: Enabling functional genomics with genome engineering
- Isaac B. Hilton and
- Charles A. Gersbach
Genome Res. October 2015 25: 1442-1455; doi:10.1101/gr.190124.115
...proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances...
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Research: Multi-species analysis of inflammatory response elements reveals ancient and lineage-specific contributions of transposable elements to NF-kB binding
- Liangxi Wang,
- Tiegh Taylor,
- Kumaragurubaran Rathnakumar,
- Nadiya Khyzha,
- Minggao Liang,
- Azad Alizada,
- Laura F. Campitelli,
- Sara E. Pour,
- Zain M. Patel,
- Lina Antounians,
- Ian C. Tobias,
- Huayun Hou,
- Timothy R. Hughes,
- Sushmita Roy,
- Jennifer A. Mitchell,
- Jason E. Fish,
- and Michael D. Wilson
Genome Res. July 2025 35: 1544-1559; Published in Advance June 6, 2025, doi:10.1101/gr.280357.124
...-stimulated mutant sequence, 0.91-fold, P-value = 0.73) (Supplemental Fig. S9B).To test the function of this enhancer in its chromatin context, we deleted the genomic region in TeloHAECs using CRISPR-Cas9 (Supplemental Fig. S9C). We performed two deletion experiments: “Deletion 1” which spans from 160 bp upstream...
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Perspective: Functional assays in Drosophila facilitate classification of variants of uncertain significance associated with rare diseases
- Jung-Wan Mok,
- Shelley B. Gibson,
- Haley A. Dostalik,
- and Shinya Yamamoto
Genome Res. July 2025 35: 1473-1484; Published in Advance June 4, 2025, doi:10.1101/gr.278291.123
...approach to studying the human variant using this analogous variant strategy is to generate a knock-in allele by directly targeting the fly ortholog gene locus using gene editing technology such as CRISPR-Cas9. This approach is particularly beneficial when: (1) the gene is dosage-sensitive, so maintaining...

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