Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System
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 prerequisite3,4,5. Since engineered nucleases used in genome editing manifest diverse efficiencies at different targeted loci1, a pre-selection of candidate sgRNA targets is necessary in order to save time and effort6,7,8,9. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing3,10. Here we use this reporter system to choose a preferred CRISPR sgRNA target from different candidate sgRNA vectors designed for specific gene editing. The protocol stated here has been implemented in our group and collaborating laboratories for the last few years to generate and evaluate CRISPR sgRNAs.
The following protocol sums up how to design suitable sgRNA through network software. Once the suitable sgRNAs are selected, we describe the different steps to obtain the required oligonucleotides as well as the approach for inserting the paired oligonucleotides into the pX330-xCas9 expression vector. We also present a method for assembling sgRNA-expressing and dual luciferase reporter vectors based on the ligation of these sequences into a predigested expression vector (steps 2-10, Figure 1A). Finally we describe how to analyze the the DNA cutting efficiency for each of the sgRNAs (steps 11-12).
Protocol
Representative Results
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.
<p class="jove_con…Declarações
The authors have nothing to disclose.
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).
Materials
| A new generation of full touch screen gradient PCR instrument | LongGene | A200 | Target gene amplification |
| AscI restriction enzymes | New England Biolabs | R0558V | Cutting target vectors |
| BbsI restriction enzyme | New England Biolabs | R0539S | Cutting target vectors |
| Clean workbench | AIRTECH | SW-CJ-2FD/VS-1300L-U | A partial purification device in the form of a vertical laminar flow, which creates a local high clean air environment |
| DH5α Competent Cells | TaKaRa | K613 | Plasmid vector transformation |
| Dual-Luciferas Reporter Assay System | Promega | E1910 | Dual-luciferas reporter assay |
| Electric thermostatic water bath | Sanfa Scientific Instruments | DK-S24 | Heating reagent by constant temperature in water bath |
| Electrophoresis | Beijing Liuyi Biotechnology Co., Ltd. | DYY-6C | Control voltage, current, etc. |
| Eppendorf Reference 2 | Eppendorf China Ltd. | Reference 2 | Accurately draw and transfer traces of liquid |
| Gel imaging analyzer | Beijing Liuyi Biotechnology Co., Ltd. | WD-9413B | For the analysis of electrophoresis gel images |
| GloMax 20/20 Luminometer | Promega | E5311 | Detect dual luciferase activity |
| High speed refrigerated centrifuge | BMH | sigma 3K15 | Nucleic acid extraction and purification |
| Intelligent biochemical incubator | Sanfa Scientific Instruments | SHP-160 | Provide a suitable temperature environment for the enzyme digestion experiment |
| LB Broth Agar | Sangon Biotech | A507003-0250 | For the cultivation of E.coli |
| Lipofectamine 3000 Transfection Reagent Kit | Thermo Fisher | L3000015 | DNA Transfection |
| SalI restriction enzymes | New England Biolabs | R3138V | Cutting target vectors |
| SanPrep Column DNA Gel Extraction Kit | Sangon Biotech | B518131-0050 | Recycling DNA fragments |
| SanPrep Column Plasmid Mini-Preps Kit | Sangon Biotech | B518191-0100 | Extraction of plasmid DNA |
| T4 DNA Ligase | New England Biolabs | M0202V | Link DNA fragment |
| TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 | TaKaRa | 9761 | DNA purification |
| Vertical pressure steam sterilizer | JIBIMED | LS-50LD | High temperature and autoclave to kill bacteria, fungi and other microorganisms in laboratory equipment |
| Water bath thermostat | Changzhou Guoyu Instrument Manufacturing Co., Ltd. | SHZ-82 | Let the bacteria keep shaking, which is good for contact with air. |
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