Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses
在苔藓中使用RNA引导的内切核酸酶进行定向诱变
发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2359 浏览次数: 13560
评审: Dennis NürnbergVinay PanwarAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2016
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Abstract
RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.
Keywords: Genome editing (基因组编辑)
RNA-guided endonucleases (RNA引导的内切核酸酶)
CRISPR/Cas system (CRISPR/Cas系统)
Targeted mutagenesis (定向诱变)
Mosses (苔藓)
Background
Targeted mutagenesis using RNA-guided endonucleases (RGENs) derived from the adaptive immune system, using the bacterial CRISPR (clusters of regularly interspaced palindromic repeats)/Cas (CRISPR-associated) systems, has dramatically advanced in recent years. In this method, the Cas9 endonuclease, derived from Streptococcus pyogenes, and an artificially designed single-chain guide RNA (sgRNA) are used. The Cas9-sgRNA complex recognizes the protospacer-adjacent motif (5’-NGG-3’) and cleaves 3 bp upstream of the target site (Jinek et al., 2012). Subsequently, random insertion and/or deletion mutations occur during the repair process for double-strand breaks (DSBs) in the DNA. As targeted mutagenesis using these RGENs is efficient as well as cost- and time-effective, it has been used for genome editing in various organisms, including many plant species. Here, we established a protocol for targeted mutagenesis using RGENs in mosses, and demonstrated it in a model and a non-model species (Nomura et al., 2016).
Materials and Reagents
- Washed and autoclaved cellophane (FUTAMURA CHEMICAL, catalog number: PS-1 )
- 9 cm plastic Petri dish (SANSEI MEDICAL, catalog number: 01-013 )
- 1.5 ml plastic tubes (FUKAEKASEI and WATSON, catalog number: 131-415C )
- 15 ml plastic tubes (FUKAEKASEI and WATSON, catalog number: 1332-015S )
- PCR tubes (NIPPON Genetics, FastGene, catalog number: FG-028DC )
- 10 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 123R-254CS )
- 200 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 123R-755CS )
- 1,000 µl plastic pipette tips (FUKAEKASEI and WATSON, catalog number: 122-804B )
- Protonemata of Physcomitrella patens (or other mosses)
- pSCgRNA, pSCOE1-fcoCas9 (Nomura et al., 2016, Figure 1A)
- DH5α-competent cells (Home-made)
- NEBuffer 2.1 (New England Biolabs, catalog number: R0539S )
- BbsI (New England Biolabs, catalog number: R0539S )
- Sterile Milli-Q water
- GEL/PCR Purification Mini Kit (Favorgen Biotech, catalog number: FAGCK 001-1 )
- Ligation high ver.2 (TOYOBO, catalog number: LGK-201 )
- ScU6p Ins. check F primer 5’-GAGGATCACGGTGTCACATGTCC-3’
- Quick Taq® HS DyeMix (TOYOBO, catalog number: DTM-101 )
- ScU6p Seq. check F primer 5’-ATGTCAAACATAACCTGG-3’
- Ampicillin (NACALAI TESQUE, catalog number: 02739-74 )
- Plasmid DNA Extraction Mini Kit (Favorgen Biotech, catalog number: FAPDE 001-1 )
- Agarose S (NIPPON GENE, catalog number: 312-01193 )
- DNA ladder markers (SMOBIO Technology, catalog number: DM3100 )
- NucleoBond® Xtra Midi (MACHEREY-NAGEL, catalog number: 740410.50 )
- G418 Disulfate (NACALAI TESQUE, catalog number: 16512-81 )
- BCDAT medium (Nishiyama et al., 2000)
- Glucose (Wako Pure Chemical Industries, catalog number: 049-31165 )
- Tks Gflex DNA polymerase (Takara Bio, catalog number: R060A )
- Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific, Invitrogen, catalog number: K275020 )
- CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1231 )
- LB agar, Miller (BD, DifcoTM, catalog number: 244520 )
- LB broth, Miller (BD, DifcoTM, catalog number: 244620 )
- Tris (hydroxymethyl) aminomethane (NACALAI TESQUE, catalog number: 35434-21 )
- Potassium chloride (Wako Pure Chemical Industries, catalog number: 163-03545 )
- Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
- Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
- Ethidium bromide solution (NACALAI TESQUE, catalog number: 14631-94 )
- LB agar plate with 50 µg/ml ampicillin (see Recipes)
- Lysis buffer (see Recipes)
- 50 x TAE buffer (see Recipes)
- 1x TAE buffer (see Recipes)
Equipment
- Plant growth chamber (SANYO, model: MLR-350HT )
- Micropipettes (Eppendorf, model: Reference® 2 )
- Sterile tweezers (Electron Microscopy Sciences, DUMONT, catalog number: 0108-5-PO )
- Thermal cycler (PCR Thermal Cycler Dice® Gradient, Takara Bio, model: TP600 )
- High-speed centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
- Block incubator (ASTEC, model: Bl-515A )
- Spectrophotometer (GE Healthcare, model: NanoVue )
- Autoclave (TOMY DIGITAL BIOLOGY, model: SX300 )
- Agarose gel electrophoresis equipment (Cosmo Bio, model: i-MyRun.N )
- Ultraviolet transilluminator (ATTO, models: DTB-20MP , TYPE-CX )
- Heated incubator (SANYO, model: MIR-262 )
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Nomura, T. and Sakakibara, H. (2017). Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses. Bio-protocol 7(12): e2359. DOI: 10.21769/BioProtoc.2359.
Download Citation in RIS Format
分类
植物科学 > 植物分子生物学 > DNA > 诱/突变
分子生物学 > DNA > 诱/突变
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