CRISPR-Cas9 Mediated Genome Editing in Drosophila
果蝇中CRISPR-Cas9介导的基因组编辑
发布: 2019年01月20日第9卷第2期 DOI: 10.21769/BioProtoc.3141 浏览次数: 10182
评审: Gal HaimovichAnonymous reviewer(s)
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The authors used this protocol in:
Nov 2013
Abstract
In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.
Keywords: Genome editing (基因组编辑)
CRISPR (CRISPR)
Cas9 (Cas9)
Drosophila (果蝇)
Heritable mutations (遗传突变)
Background
CRISPR-Cas9, an acquired immune system derived from bacteria, is made up of a single-stranded homing RNA and a Cas9 protein that has the properties of endonuclease activity (Barrangou et al., 2007; Gasiunas et al., 2012). These two elements can form a complex where the sgRNA guides the Cas9 protein to the specific DNA sequences, resulting in the double-stranded DNA break. Compared with the traditional gene editing system like ZFN and TALEN, the efficient CRISPR-Cas9 system is simpler to design and easier to manipulate, which greatly expands its applications.
Drosophila is one of the best model organisms favored by biomedical researchers in various fields that benefit from the advanced genetic tools. Here we describe how to apply the CRISPR-Cas9 system to generate gene-targeted mutations in Drosophila. The specificity of the target is determined by the 20 nt sequence within the sgRNA, which is transcribed under the control of U6b promoter and then recruits the Cas9 protein which is driven by nanos regulator sequence (Ren et al., 2013). In this case, we only need to select sgRNA of the target gene and then clone it into the pU6B plasmid. After plasmid preparation and purification, we microinject it into the embryos of transgenic Cas9 flies, from whose offspring we can get heritable mutations.
Materials and Reagents
- 1.5 ml MaxyClear Microtubes (Axygen, catalog number: MCT-150-C)
- 0.2 ml Polypropylene PCR Tube (Axygen, catalog number: PCR-0208-C)
- Pipette tips (Corning, Axygen®)
- 0.22 μm Millipore filters (Millipore, catalog number: R7MA69670)
- Fly stocks (All Drosophila strains are stored in Tsinghua Fly Center)
- y,v; attp40{nos-Cas9} (TH00788)
- y,v;; attp2{nos-Cas9} (TH00787)
- y sc v (TB00077)
- y sc v; Gla Bc/CyO (TB00023)
- y sc v;; Dr,e/TM3,Sb (TB00139)
- Trans5α Chemically Competent Cell (Transgen Biotech, catalog number: CD201-01)
- PCR primers (Table 1), custom-synthesized
Table 1. The primers for colony PCR and DNA sequencing
- pU6B (Ampicillin resistant, Figure 1)
Figure 1. The construct map of pU6B. The insertion fragment between two BbsI sites is the marker to detect whether sgRNA is ligated with the vector. U6Bp-FS1 and V20-R are the primers for colony PCR and DNA sequencing. - BbsI (New England Biolabs, catalog number: R0539L)
- GoTag Green Master Mix, 2x (Promega, catalog number: 000179370)
- T4 DNA ligase (New England Biolabs, catalog number: M0202L)
- AxyPrep Plasmid Miniprep Kit (Corning, Axygen®, catalog number: AP-MN-P-250)
- AxyPrep DNA Gel Extraction Kit (Corning, Axygen®, catalog number: AP-GX-250)
- PurePlasmid Mini Kit (CWBiotech, catalog number: CW0500M)
- Binding Buffer PB (QIAGEN, catalog number: 154051779)
- Phenol-chloroform (Solarbio, catalog number: P1012)
- Ethanol absolute (Sigma, catalog number: SHBC3572V)
- Isopropyl alcohol (Sigma, catalog number: SZBC2260V)
- Sodium acetate (Amrosco, catalog number: 1123C206)
- 10x PBS (Double Helix, catalog number: S0905A)
- 50x TAE (Double Helix, catalog number: P0309A)
- Agarose (Invitrogen, catalog number: 0000602315)
- LB powder (BD, catalog number: 2171199)
- KCl (AMRESCO, catalog number: 7447-40-7)
- Tris-HCl (AMRESCO, catalog number: 1185-53-1)
- EDTA (AMRESCO, catalog number: 60-00-4)
- NaCl (AMRESCO, catalog number: 7647-14-5)
- SDS (Beyotime, catalog number: ST628)
- Proteinase K (Roche, catalog number: P8601-100)
- Ampicillin (Solarbio, catalog number: A8180)
- Ethidium bromide (Sigma, catalog number: E8751)
- 0.2 M Sodium phosphate buffer stock solution (pH 6.8) (see Recipes)
- 0.2 M Na2HPO4 stock solution (see Recipes)
- 0.2 M NaH2PO4 stock solution (see Recipes)
- 10x Injection Buffer (see Recipes)
- 10x Annealing Buffer (see Recipes)
- 1x PBS (see Recipes)
- LB (Luria Bertani) medium (see Recipes)
- 1x TAE (see Recipes)
- Lysis Buffer (see Recipes)
- 1.5% agarose gel (see Recipes)
Equipment
- Pipette (Eppendorf)
- Microwave (SANYO, model: EM-2509EB1)
- Autoclave (SANYO, model: MLS-3780)
- PCR thermal cycler (Eppendorf, Mastercycler nexus GSX1)
- Refrigerated centrifuge (Eppendorf, model: 5417R)
- DNA electrophoresis apparatus (Bio-Rad)
- Microscope (Olympus, model: SZX10)
- NanoDrop 2000 Spectrophotometer (Thermo Scientific)
- Incubator (GINGKO)
- 4 °C refrigerator (Haier)
- -20 °C freezer (Haier)
Procedure
文章信息
版权信息
© 2019 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Peng, P., Wang, X., Shen, D., Sun, J., Jia, Y., Xu, R., Zhu, L. and Ni, J. (2019). CRISPR-Cas9 Mediated Genome Editing in Drosophila. Bio-protocol 9(2): e3141. DOI: 10.21769/BioProtoc.3141.
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分类
分子生物学 > DNA > DNA 修饰
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