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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
...-scale mutagenesis to other nonmodel organisms.ResultsDesign and construction of the CRISPR single-guide RNA libraryBased on our previously reported, highly efficient, CRISPR-Cas9-mediated B. mori targeted mutagenesis in the whole animal and the pooled library in cultured cells (Ma et al. 2014a; Chang et...
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Method: A versatile type VI CRISPR-based approach for targeted m⁶A demethylation in mRNAs
- Panagiotis G. Adamopoulos,
- Konstantina Athanasopoulou,
- and Andreas Scorilas
Genome Res. January 2026 36: 169-182; Published in Advance December 11, 2025, doi:10.1101/gr.280476.125
...information on a target site of interest.The advent of innovative methodologies, such as those integrating CRISPR/Cas systems with RNA-processing approaches, has expedited research in this domain, furnishing novel tools for dissecting the roles of post-transcriptional modifications in gene expression dynamics...
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Research: APOBEC3A drives deaminase mutagenesis in human gastric epithelium
- Yohan An,
- Ji-Hyun Lee,
- Joonoh Lim,
- Jeonghwan Youk,
- Seongyeol Park,
- Ji-Hyung Park,
- Kijong Yi,
- Taewoo Kim,
- Chang Hyun Nam,
- Won Hee Lee,
- Soo A Oh,
- Yoo Jin Bae,
- Thomas M. Klompstra,
- Haeun Lee,
- Jinju Han,
- Junehwak Lee,
- Jung Woo Park,
- Jie-Hyun Kim,
- Hyunki Kim,
- Hugo Snippert,
- Bon-Kyoung Koo,
- and Young Seok Ju
Genome Res. October 2025 35: 2158-2172; Published in Advance August 26, 2025, doi:10.1101/gr.280338.124
...Technology, #07922) (see Supplemental Methods; Supplemental Table S11).Preparation of vectors for transfectionTwo vectors were purchased: (1) CMV-rtTA-HygR vector (Addgene, #102423) and (2) CRISPR-Cas9 vectors containing gRNA sequence for TP53 (Addgene, #121917). To generate the pPB-CMVmin-APOBEC (A3A or A3B...
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Method: High-fidelity, large-scale targeted profiling of microsatellites
- Caitlin A. Loh,
- Danielle A. Shields,
- Adam Schwing,
- and Gilad D. Evrony
Genome Res. July 2024 34: 1008-1026; Published in Advance July 16, 2024, doi:10.1101/gr.278785.123
...-scale panel of human microsatellites for targeted profiling (Fig. 1A).View larger version: In this window In a new window Figure 1. Overview of method and probe design strategy. (A) Schematic of microsatellite panel design, library preparation, hybridization capture, sequencing, and analysis. (B) The three...
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Review: Leveraging the power of long reads for targeted sequencing
- Shruti V. Iyer,
- Sara Goodwin,
- and William Richard McCombie
Genome Res. November 2024 34: 1701-1718; doi:10.1101/gr.279168.124
...fragment length via hybridization, and it is extremely difficult, if not impossible, to accurately assemble DNA fragments <10 kb in regions like this.CRISPR-Cas9-based enzymatic targeting approachesThe CRISPR–Cas system provides a specific, rapid, and flexible enzymology (Jinek et al. 2012; Doudna...
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Method: Systematic mapping of TF-mediated cell fate changes by a pooled induction coupled with scRNA-seq and multi-omics approaches
- Muyoung Lee,
- Qingqing Guo,
- Mijeong Kim,
- Joonhyuk Choi,
- Alia Segura,
- Alper Genceroglu,
- Lucy LeBlanc,
- Nereida Ramirez,
- Yu Jin Jang,
- Yeejin Jang,
- Bum-Kyu Lee,
- Edward M. Marcotte,
- and Jonghwan Kim
Genome Res. March 2024 34: 484-497; Published in Advance April 5, 2024, doi:10.1101/gr.277926.123
..., CRISPR-Cas9-mediated perturbation, and overexpression screens, to address numerous questions in biology and medicine without relying on a limited number of reporter genes (Adamson et al. 2016; Dixit et al. 2016; Jaitin et al. 2016; Aarts et al. 2017; Datlinger et al. 2017).Here, we introduce i...
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Research: Genetic inhibitors of APOBEC3B-induced mutagenesis
- Tony M. Mertz,
- Elizabeth Rice-Reynolds,
- Ly Nguyen,
- Anna Wood,
- Cameron Cordero,
- Nicholas Bray,
- Victoria Harcy,
- Rudri K. Vyas,
- Debra Mitchell,
- Kirill Lobachev,
- and Steven A. Roberts
Genome Res. September 2023 33: 1568-1581; Published in Advance August 2, 2023, doi:10.1101/gr.277430.122
...-induced mutagenesis. We created a library of haploid yeast deletion strains expressing A3B using a mating, sporulation, and selection strategy (Fig. 1A). We identified ORF deletions (screen hits) that increased the frequency of canavanine-resistant (CanR) colonies at least twofold in each of three successive rounds...
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Method: Genome-scale identification of cellular pathways required for cell surface recognition
- Sumana Sharma,
- S. Josefin Bartholdson,
- Amalie C.M. Couch,
- Kosuke Yusa,
- and Gavin J. Wright
Genome Res. September 2018 28: 1372-1382; Published in Advance June 18, 2018, doi:10.1101/gr.231183.117
...using highly efficient CRISPR methods now presents the possibility to interrogate the genetic basis of cellular phenotypes on a -wide scale (Koike-Yusa et al. 2014; Shalem et al. 2014, 2015; Wang et al. 2014). Libraries of cells that contain biallelic targeted loss-of-function alleles can be created...
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Method: Beta-binomial modeling of CRISPR pooled screen data identifies target genes with greater sensitivity and fewer false negatives
- Hyun-Hwan Jeong,
- Seon Young Kim,
- Maxime W.C. Rousseaux,
- Huda Y. Zoghbi,
- and Zhandong Liu
Genome Res. June 2019 29: 999-1008; Published in Advance April 23, 2019, doi:10.1101/gr.245571.118
...interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) systems have propelled pooled screen approaches into even wider use. Besides the relative simplicity and low cost, the robustness of hit identification has reduced the requirement for redundancy in the number of targeting...
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Method: Single-cell Rapid Capture Hybridization sequencing reliably detects isoform usage and coding mutations in targeted genes
- Hongke Peng,
- Jafar S. Jabbari,
- Luyi Tian,
- Changqing Wang,
- Yupei You,
- Chong Chyn Chua,
- Natasha S. Anstee,
- Noorul Amin,
- Andrew H. Wei,
- Nadia M. Davidson,
- Andrew W. Roberts,
- David C.S. Huang,
- Matthew E. Ritchie,
- and Rachel Thijssen
Genome Res. April 2025 35: 942-955; Published in Advance January 10, 2025, doi:10.1101/gr.279322.124
....6× beads. For the BTK and SF3B1 gene capture, 21 samples were pooled together and sequenced on PromethION R9.4.1 flow cell (Fig. 1).The scRaCH-seq libraries can also be sequenced on a PromethION R10.4.1 flow cell and using the SQK-NBD114.24 Native Barcoding Kit 24 to index the samples and pool them...
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