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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Abstract

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Introduction

The CRISPR sgRNAs comprise a 20-nucleotide sequence (the protospacer), which is complementary to the genomic target sequence1,2. Although highly efficient, the ability of the CRISPR/Cas system to modify a given genomic site requires the generation of a vector carrying an efficient sgRNA unique to the target site(s)2. This paper describes the key steps in the generation of that sgRNA vector.

For successful genome editing using the CRISPR/Cas system, the use of highly efficient sgRNAs is a crucial prerequisite3,

Protocol

1. sgRNA oligonucleotide design

  1. Design sgRNAs using online tools such as the Cas-Designer online tool (http://www.rgenome.net/cas-designer/). The PAM sequence is important based on the Cas9 being used. For xCas9, the relevant PAM sequences are NG and the former referred Cas-Designer online tool can generate xCas9 relevant sgRNAs.
    1. Use sgRNA design tools that encompass algorithms for on- and off-target prediction (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design)

Representative Results

The methods outlined in this protocol are for the construction of sgRNA and xCas9 expression vectors and then for the optimization screening of sgRNA oligos with relatively higher gene targeting efficiencies. Here we display a representative example of 3 sgRNA targets to sheep DKK2 exon 1. SgRNA and xCas9 expressing vectors can be built by predigesting the vector backbone (Figure 2) followed by ligating it in a series of short double-strand DNA fragments through annealing oligo pair.......

Discussion

The sgRNA vector cloning procedures we have described here facilitates efficient production of sgRNAs, with most of the costs derived from the oligonucleotide ordering and vector sequencing. While the outlined method is designed to allow users to generate sgRNAs for use with CRISPR/Cas9, the protocol can easily be adapted for use with Cas9 orthologues or other RNA-guided endonucleases such as Cpf1, introducing minor modifications to the vector backbone and the oligonucleotide overhanging sequences.

Acknowledgements

This project was funded by First Class Grassland Science Discipline Program of Shandong Province (China), National Natural Science Foundation of China (31301936, 31572383), the Special Fund for Agro-scientific Research in the Public Interest (201403071), National risk assessment major special project of milk product quality and safety (GJFP201800804) and Projects of Qingdao People's Livelihood Science and Technology (19-6-1-68-nsh, 14-2-3-45-nsh, 13-1-3-88-nsh).

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Materials

NameCompanyCatalog NumberComments
A new generation of full touch screen gradient PCR instrumentLongGeneA200Target gene amplification
AscI restriction enzymesNew England BiolabsR0558VCutting target vectors
BbsI restriction enzymeNew England BiolabsR0539SCutting target vectors
Clean workbenchAIRTECHSW-CJ-2FD/VS-1300L-UA partial purification device in the form of a vertical laminar flow, which creates a local high clean air environment
DH5α Competent CellsTaKaRaK613Plasmid vector transformation
Dual-Luciferas Reporter Assay SystemPromegaE1910Dual-luciferas reporter assay
Electric thermostatic water bathSanfa Scientific InstrumentsDK-S24Heating reagent by constant temperature in water bath
ElectrophoresisBeijing Liuyi Biotechnology Co., Ltd.DYY-6CControl voltage, current, etc.
Eppendorf Reference 2Eppendorf China Ltd.Reference 2Accurately draw and transfer traces of liquid
Gel imaging analyzerBeijing Liuyi Biotechnology Co., Ltd.WD-9413BFor the analysis of electrophoresis gel images
GloMax 20/20 LuminometerPromegaE5311Detect dual luciferase activity
High speed refrigerated centrifugeBMHsigma 3K15Nucleic acid extraction and purification
Intelligent biochemical incubatorSanfa Scientific InstrumentsSHP-160Provide a suitable temperature environment for the enzyme digestion experiment
LB Broth AgarSangon BiotechA507003-0250For the cultivation of E.coli
Lipofectamine 3000 Transfection Reagent KitThermo FisherL3000015DNA Transfection
SalI restriction enzymesNew England BiolabsR3138VCutting target vectors
SanPrep Column DNA Gel Extraction KitSangon BiotechB518131-0050Recycling DNA fragments
SanPrep Column Plasmid Mini-Preps KitSangon BiotechB518191-0100Extraction of plasmid DNA
T4 DNA LigaseNew England BiolabsM0202VLink DNA fragment
TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0TaKaRa9761DNA purification
Vertical pressure steam sterilizerJIBIMEDLS-50LDHigh temperature and autoclave to kill bacteria, fungi and other microorganisms in laboratory equipment
Water bath thermostatChangzhou Guoyu Instrument Manufacturing Co., Ltd.SHZ-82Let the bacteria keep shaking, which is good for contact with air.

References

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CRISPRPlasmid ConstructionDual Luciferase Reporter SystemSgRNAGene EditingPCRCell TransformationBacterial ColonyRecombinant PlasmidsCell LysisDual Luciferase Assay

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