在苔藓中使用RNA引导的内切核酸酶进行定向诱变

Toshihisa  Nomura; Hitoshi  Sakakibara

doi:10.21769/BioProtoc.2359

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Peer-reviewed

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

TN Toshihisa Nomura email

HS Hitoshi Sakakibara

发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2359 浏览次数: 12198

评审: Dennis NürnbergVinay PanwarAnonymous reviewer(s)

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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 Scientific^TM, catalog number: K1231 )
LB agar, Miller (BD, Difco^TM, catalog number: 244520 )
LB broth, Miller (BD, Difco^TM, 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

English

中文翻译

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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.