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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
...various organisms (Drosophila, zebrafish, sea squirt, worms, and cell culture cells), which determined sgRNA cleavage efficiency for their performance using the PlatinumCRISPr design tool (Doench et al. 2014, 2016; Gagnon et al. 2014; Ren et al. 2014; Wang et al. 2014; Chari et al. 2015; Farboud and Meyer...
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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
...markers, respectively (Ma et al. 2014a; Xu et al. 2017), and knockout was achieved by crossing them (Fig. 1A).View larger version: In this window In a new window Figure 1. Design and construction of the CRISPR single-guide RNA (sgRNA) library. (A) The strategy of the mutant library construction. (B...
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Method: Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore
- Narendra Chaudhary,
- Si-Hyeong Nho,
- Hayoon Cho,
- Narangerel Gantumur,
- Jae Sun Ra,
- Kyungjae Myung,
- and Hajin Kim
Genome Res. September 2020 30: 1306-1316; Published in Advance September 4, 2020, doi:10.1101/gr.260018.119
...compared between the fluorophore designs in C before and after colony picking (n > 200 cells). (E) Fluorescence images of fixed stable cell lines with the SunTag split-sfGFP CRISPR-dCas9 system in polyclonal and clonal after transient transfection with sgRNA-12 × MBS targeting a pericentromeric region...
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Resource: Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library
- Jianjie He,
- Kai Zhang,
- Shuxiang Yan,
- Mi Tang,
- Weixian Zhou,
- Yongtai Yin,
- Kang Chen,
- Chunyu Zhang,
- and Maoteng Li
Genome Res. May 2023 33: 798-809; Published in Advance June 8, 2023, doi:10.1101/gr.277650.123
...in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve -scale targeted editing in an allotetraploid crop of Brassica napus. A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088...
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Method: CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes
- Marzia Rossato,
- Luca Marcolungo,
- Luca De Antoni,
- Giulia Lopatriello,
- Elisa Bellucci,
- Gaia Cortinovis,
- Giulia Frascarelli,
- Laura Nanni,
- Elena Bitocchi,
- Valerio Di Vittori,
- Leonardo Vincenzi,
- Filippo Lucchini,
- Kirstin E. Bett,
- Larissa Ramsay,
- David James Konkin,
- Massimo Delledonne,
- and Roberto Papa
Genome Res. May 2023 33: 787-797; Published in Advance May 1, 2023, doi:10.1101/gr.277628.122
.... Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34: 184–191. doi:10.1038/nbt.3437 ↵Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE. 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity...
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Method: CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs
- Ho Joung Lee,
- Hyun Ju Kim,
- and Sang Jun Lee
Genome Res. May 2020 30: 768-775; Published in Advance April 23, 2020, doi:10.1101/gr.257493.119
...edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial s.The function of bacterial clustered regularly interspaced short palindromic repeats (CRISPR) is to “memorize” certain DNA sequences derived from...
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Method: JACKS: joint analysis of CRISPR/Cas9 knockout screens
- Felicity Allen,
- Fiona Behan,
- Anton Khodak,
- Francesco Iorio,
- Kosuke Yusa,
- Mathew Garnett,
- and Leopold Parts
Genome Res. March 2019 29: 464-471; Published in Advance January 23, 2019, doi:10.1101/gr.238923.118
...effect improves specificity of CRISPR Cas9 essentiality screens in cancer cells. Nat Genet 49: 1779–1784. doi:10.1038/ng.3984 ↵Moreno-Mateos MA, Vejnar CE, Jd B, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ. 2015. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat...
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Method: Genome-wide CRISPR screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori
- Jiasong Chang,
- Ruolin Wang,
- Kai Yu,
- Tong Zhang,
- Xiaoxu Chen,
- Yue Liu,
- Run Shi,
- Xiaogang Wang,
- Qingyou Xia,
- and Sanyuan Ma
Genome Res. May 2020 30: 757-767; Published in Advance May 18, 2020, doi:10.1101/gr.249045.119
...E-Mi-EGFP cells.To test the knockout efficiency of pB-CRISPR, we first established a cell line that stably expressed enhanced green fluorescent protein (EGFP; BmE-EGFP) using a second general transposon, Minos (Supplemental Fig. S1A,C; Supplemental Materials). Three sgRNAs targeting EGFP were designed...
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Method: CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans
- Ping Wei,
- Wen Xue,
- Yun Zhao,
- Guang Ning,
- and Jiwu Wang
Genome Res. January 2020 30: 95-106; Published in Advance November 13, 2019, doi:10.1101/gr.250811.119
...-mediated integration into the attP2 landing site, and the UAS-cDNA/ORF constructs used for transgenes were described in Supplemental Table S1.sgRNA preparationTo design the sgRNAs used for cleavage of vector backbones of cDNA/ORF plasmids, we used Optimized CRISPR Design (http://crispr.mit.edu) to search...
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Method: DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA
- Daesik Kim and
- Jin-Soo Kim
Genome Res. December 2018 28: 1894-1900; Published in Advance November 9, 2018, doi:10.1101/gr.236620.118
...engineering via Cas9. Science 339: 823–826. doi:10.1126/science.1232033 ↵Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ. 2015. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12: 982–988. doi:10.1038/nmeth.3543 ↵Park J, Lim K...
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