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A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
Published: Vol 11, Iss 7, Apr 5, 2021 DOI: 10.21769/BioProtoc.3971 Views: 4912
Edited by: Alessandro Didonna Reviewed by: Kangquan YinJonathan R Gilkerson
Original research article
The authors used this protocol in:
Jan 2020
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Abstract
CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.
Keywords: Cloning system
Multiplex PCR
CRISPR/Cas9
Genome editing
Rice
Background
Bacteria defend against viruses through a protein system, consisting of the clustered regularly interspaced short palindromic repeat (CRISPR), the CRISPR-associated (Cas) protein, CRISPR RNAs (crRNAs) and trans-encoded crRNA (tracrRNA). Researchers have now developed this system into a key tool for targeted genome editing. CRISPR – binary vectors express two elements – the sgRNAs with a target sequence (target-sgRNAs) and Cas9 protein – to cleave target genomic regions. Feng et al. (2013) have constructed gateway vectors to co-express Cas9 and sgRNAs in plants through Agrobacterium sp.-mediated transformation. In a restriction-ligation reaction, a gene-specific sgRNA spacer substitutes the target region in the entry clone, which encodes attL recombination sites. Then, an “LR Clonase” reaction transfers the target-sgRNA cassette into a destination clone, which contains a Cas9 expression cassette (Feng et al., 2013). Ma et al. (2016) have developed this basic system into a CRISPR/Cas9 system for multiplex genome editing in rice. In this approach, a restriction-ligation reaction inserts a spacer into intermediate vectors to produce an sgRNA expression cassette, which fuses with adaptors for Golden Gate cloning or Gibson Assembly (Ma et al., 2016). In an alternative approach, an overlapping PCR, with two rounds of reactions, can also establish a sgRNA expression cassette with adaptors. The sgRNA expression cassette can then be introduced into a binary vector via Golden Gate cloning or Gibson Assembly (Ma et al., 2016). However, traditional cloning, based on restriction-ligation reactions or two-round overlapping PCRs, is time-consuming.
Herein, we report a novel method to construct the binary vectors with one or two targets by a single round of PCR and a single LR reaction or Golden Gate cloning (Liu et al., 2020). Using this system, an expression clone can be constructed within 36 hours, which significantly improves efficiency and reduces costs.
Materials and Reagents
200 µl PCR tubes (Biosharp, catalog number: BS-02-P )
1.5 ml microcentrifuge tubes (Biosharp, catalog number: BS-15-M )
Pipette tips (Biosharp, catalog numbers: BS-10-T , BS-200-T , BS-1000-T )
Competent E. coli T1 cells (TransGen, catalog number: CD501-02 )
LR clonase (GatewayTM LR ClonaseTM Enzyme Mix, catalog number: 11791-043 )
NEB Cutsmart buffer (New England BioLabs, catalog number: B7204S )
NEBuffer 3.1 (New England BioLabs, catalog number: B7203S )
EcoRV (New England BioLabs, catalog number: R0195L )
BsaI-HF (New England BioLabs, catalog number: R3733L )
T4 DNA ligase (New England BioLabs, catalog number: M0202L )
PCR SuperMix (TransGen, catalog number: AS111-11 )
KOD FX (Toyobo, catalog number: KFX-101 )
dNTPs Mixture (2mM) (Toyobo, catalog number: NTP-201 )
Sterilized double distilled H2O (Phygene, catalog number: PH0727 )
DreamTaq or other equivalent DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K1072 )
Agarose (Biowest, catalog number: 111860 )
Plasmid prep mini kit (OMEGA, catalog number: D3350-01 )
Gel Extraction Kit (OMEGA, catalog number: D2500-01 )
Spectinomycin (Sigma-Aldrich, catalog number: PHR1441 )
Tris (Solarbio Life Scientific, catalog number: T8060 )
Acetic acid (MERCK, catalog number: M10006307 )
0.5 M EDTA (Solarbio Life Scientific, catalog number: E1170 )
Tryptone (Oxoid, catalog number: LP0042 )
Yeast extract (Oxoid, catalog number: LP0021 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
Ethidium bromide (EB) (Sigma-Aldrich, catalog number: E8751 )
Universal primers for PCR screening (see Table 1)
Table 1. Universal primers
Primers Sequences (5′ → 3′) OJP001 TCGCGTTAACGCTAGCATGGATCTC OJP002 GTAACATCAGAGATTTTGAGACAC OJP008 ACCACCTCGGCTATCCACA OJP026 ATAGCCTTATGCAGTTGCTCT OJP065 CGACTCGGTGCCACTTTTTC PJF997 (the donor vector containing OsU3-sgRNA expression cassette), PJF999 (the donor vector containing OsU6-sgRNA expression cassette), PJG090 (the donor vector for amplification of two spacers), PJG097 (the destination vector for one target), PJG112 (the destination vector for two targets). All the vectors were developed in our previous work (Liu et al., 2020)
50× TAE electrophoresis buffer (see Recipes)
LB medium (see Recipes)
LB agar medium (see Recipes)
Equipment
Pipettes (Eppendorf)
Microcentrifuge (Eppendorf, model: Centrifuge 5424 )
Heating block (Hangzhou Allsheng Instruments, model: MK-20 )
Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: Veriti® 96 well thermal cycler )
Water bath (Shanghai Binglin, model: BLHH-6N )
NanoDrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
Gel Imaging System (The ChemiDoc XRS+ System, BIO-RAD, model: 1708265 )
Software
SnapGene (GSL Biotech LLC, https://www.snapgene.com/)
WPS Excel (Kingsoft Office, https://www.wps.cn/)
Procedure
Category
Plant Science > Plant molecular biology > Genetic analysis
Molecular Biology > DNA > DNA cloning
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