基因工程大鼠和小鼠中的 CRISPR 脱靶分析。
CRISPR off-target analysis in genetically engineered rats and mice.
机构信息
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA, USA.
Jack Baskin School of Engineering, University of California, Santa Cruz, Santa Cruz, CA, USA.
出版信息
Nat Methods. 2018 Jul;15(7):512-514. doi: 10.1038/s41592-018-0011-5. Epub 2018 May 21.
Abstract
Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.
摘要
尽管 CRISPR 得到了广泛应用，但关于 Cas9 介导的修饰啮齿动物中脱靶的频率和影响的综合数据仍然有限。在这里，我们展示了来自 81 个基因组编辑项目的深度测序数据，这些项目针对小鼠和大鼠基因组中的 1423 个预测脱靶位点，其中 32 个已经得到证实，并表明高保真 Cas9 版本降低了体内的脱靶突变率。使用来自十个用单向导 RNA（sgRNA）处理的小鼠胚胎及其遗传父母的全基因组测序数据，我们发现了 43 个脱靶位点，其中 30 个是通过 GUIDE-seq 的改编版本预测的。
相似文献
1
CRISPR off-target analysis in genetically engineered rats and mice.
Nat Methods. 2018 Jul;15(7):512-514. doi: 10.1038/s41592-018-0011-5. Epub 2018 May 21.
2
Off- and on-target effects of genome editing in mouse embryos.
J Reprod Dev. 2019 Feb 8;65(1):1-5. doi: 10.1262/jrd.2018-128. Epub 2018 Dec 6.
3
Genome modification by CRISPR/Cas9.
FEBS J. 2014 Dec;281(23):5186-93. doi: 10.1111/febs.13110. Epub 2014 Nov 7.
4
CRISPR/Cas9 Immune System as a Tool for Genome Engineering.
Arch Immunol Ther Exp (Warsz). 2017 Jun;65(3):233-240. doi: 10.1007/s00005-016-0427-5. Epub 2016 Oct 3.
5
Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research.
Curr Opin Plant Biol. 2016 Apr;30:70-7. doi: 10.1016/j.pbi.2016.01.007. Epub 2016 Feb 18.
6
CRISPR/Cas9 for genome editing: progress, implications and challenges.
Hum Mol Genet. 2014 Sep 15;23(R1):R40-6. doi: 10.1093/hmg/ddu125. Epub 2014 Mar 20.
7
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6.
8
Genome editing using Cas9 nickases.
Methods Enzymol. 2014;546:161-74. doi: 10.1016/B978-0-12-801185-0.00008-8.
9
AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3470-6. doi: 10.1167/iovs.16-19316.
10
Genetic screens and functional genomics using CRISPR/Cas9 technology.
FEBS J. 2015 Apr;282(8):1383-93. doi: 10.1111/febs.13248. Epub 2015 Mar 16.
引用本文的文献
1
Biosafety considerations triggered by genome-editing technologies.
Biosaf Health. 2025 May 13;7(3):141-151. doi: 10.1016/j.bsheal.2025.05.003. eCollection 2025 Jun.
2
Enhancing Specificity, Precision, Accessibility, Flexibility, and Safety to Overcome Traditional CRISPR/Cas Editing Challenges and Shape Future Innovations.
Adv Sci (Weinh). 2025 Jul;12(28):e2416331. doi: 10.1002/advs.202416331. Epub 2025 Jun 23.
3
A versatile CRISPR/Cas9 system off-target prediction tool using language model.
Commun Biol. 2025 Jun 6;8(1):882. doi: 10.1038/s42003-025-08275-6.
4
Activation of Human FPR2 with AT-RvD1 Resolves Acute Sialadenitis in Vivo.
Inflammation. 2025 Jun 4. doi: 10.1007/s10753-025-02320-6.
5
Application of CRISPR/Cas gene editing for infectious disease control in poultry.
Open Life Sci. 2025 May 20;20(1):20251095. doi: 10.1515/biol-2025-1095. eCollection 2025.
6
Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease.
Curr Protoc. 2025 Feb;5(2):e70109. doi: 10.1002/cpz1.70109.
7
A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin.
Biol Open. 2025 Mar 15;14(3). doi: 10.1242/bio.061677. Epub 2025 Mar 17.
8
CRISPRoffT: comprehensive database of CRISPR/Cas off-targets.
Nucleic Acids Res. 2025 Jan 6;53(D1):D914-D924. doi: 10.1093/nar/gkae1025.
9
VDGE: a data repository of variation database for gene-edited animals across multiple species.
Nucleic Acids Res. 2025 Jan 6;53(D1):D1250-D1260. doi: 10.1093/nar/gkae956.
10
Crispr-SGRU: Prediction of CRISPR/Cas9 Off-Target Activities with Mismatches and Indels Using Stacked BiGRU.
Int J Mol Sci. 2024 Oct 11;25(20):10945. doi: 10.3390/ijms252010945.
本文引用的文献
1
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
Nature. 2017 Oct 19;550(7676):407-410. doi: 10.1038/nature24268. Epub 2017 Sep 20.
2
Mapping the genomic landscape of CRISPR-Cas9 cleavage.
Nat Methods. 2017 Jun;14(6):600-606. doi: 10.1038/nmeth.4284. Epub 2017 May 1.
3
CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.
Nat Methods. 2017 Jun;14(6):607-614. doi: 10.1038/nmeth.4278. Epub 2017 May 1.
4
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.
Genome Biol. 2016 Jul 5;17(1):148. doi: 10.1186/s13059-016-1012-2.
5
Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.
Nature. 2016 May 5;533(7601):125-9. doi: 10.1038/nature17664. Epub 2016 Apr 27.
6
Activation of proto-oncogenes by disruption of chromosome neighborhoods.
Science. 2016 Mar 25;351(6280):1454-1458. doi: 10.1126/science.aad9024. Epub 2016 Mar 3.
7
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.
Nat Biotechnol. 2016 Feb;34(2):184-191. doi: 10.1038/nbt.3437. Epub 2016 Jan 18.
8
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6.
9
Rationally engineered Cas9 nucleases with improved specificity.
Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126/science.aad5227. Epub 2015 Dec 1.
10
Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice.
PLoS One. 2015 May 29;10(5):e0128330. doi: 10.1371/journal.pone.0128330. eCollection 2015.
Zotero 插件