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&#39;s Livelihood Science and Technology (19-6-1-68-nsh, 14-2-3-45-nsh, 13-1-3-88-nsh).

1. sgRNA oligonucleotide design
<ol>
	<li>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.
	<ol>
		<li>Use sgRNA design tools that encompass algorithms for on- and off-target prediction (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design)<sup class="xref...

<ol>
	<li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surrogate+reporter+system+for+multiplexable+evaluation+of+CRISPR/Cas9+in+targeted+mutagenesis.">A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis.</a> Scientific Reports. 8 (1), 1042 (2018).</li><li>Nageshwaran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+Guide+RNA+Cloning+for+Mammalian+Systems.">CRISPR Guide RNA Cloning for Mammalian Systems.</a> Journal of Visualized Experiments. (140), (2018).</li><li>Dang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+sgRNA+structure+to+improve+CRISPR-Cas9+knockout+efficiency.">Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.</a> Genome Biology. 16, 280 (2015).</li><li>Doench, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+sgRNA+design+to+maximize+activity+and+minimize+off-target+effects+of+CRISPR-Cas9.">Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.</a> Nature Biotechnology. 34 (2), 184-191 (2016).</li><li>Moreno-Mateos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRscan:+designing+highly+efficient+sgRNAs+for+CRISPR-Cas9+targeting+in+vivo.">CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.</a> Nature Methods. 12 (10), 982-988 (2015).</li><li>Joung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-Cas9+knockout+and+transcriptional+activation+screening.">Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.</a> Nature Protocols. 12 (4), 828-863 (2017).</li><li>Yang, L., Yang, J. L., Byrne, S., Pan, J., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-Directed+Genome+Editing+of+Cultured+Cells.">CRISPR/Cas9-Directed Genome Editing of Cultured Cells.</a> Current Protocols in Molecular Biology. 107, 1-17 (2014).</li><li>Yang, L., Mali, P., Kim-Kiselak, C., Church, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas-mediated+targeted+genome+editing+in+human+cells.">CRISPR-Cas-mediated targeted genome editing in human cells.</a> Methods in Molecular Biology. 1114, 245-267 (2014).</li><li>Vidigal, J. A., Ventura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+one-step+generation+of+paired+gRNA+CRISPR-Cas9+libraries.">Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries.</a> Nature Communications. 6, 8083 (2015).</li><li>Mazon, G., Mimitou, E. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Homologous+recombination+in+DNA+double-strand+break+repair.">SnapShot: Homologous recombination in DNA double-strand break repair.</a> Cell. 142 (4), 646 (2010).</li><li>Doench, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nature Biotechnology. 32 (12), 1262-1267 (2014).</li><li>Lee, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+CRISPR-Cas9+to+increase+its+specificity.">Directed evolution of CRISPR-Cas9 to increase its specificity.</a> Nature Communications. 9 (1), 3048 (2018).</li><li>Sung, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+gene+knockout+in+mice+and+zebrafish+with+RNA-guided+endonucleases.">Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.</a> Genome Research. 24 (1), 125-131 (2014).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154 (6), 1380-1389 (2013).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (12), 5463-5467 (1977).</li><li>Ruan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+transgene+knockin+at+the+H11+locus+in+pigs.">Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs.</a> Scientific Reports. 5, 14253 (2015).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+plasmid+with+double+fluorescent+groups+and+its+application+as+standard+substance.">An plasmid with double fluorescent groups and its application as standard substance.</a> China patent. , (2012).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+porcine+p65+subunit+of+NF-kappaB+and+its+association+with+virus+antibody+levels.">Characterization of the porcine p65 subunit of NF-kappaB and its association with virus antibody levels.</a> Molecular Immunology. 48 (6-7), 914-923 (2011).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pair+of+sgRNAs+targeting+porcine+RELA+gene.">A pair of sgRNAs targeting porcine RELA gene.</a> China patent. , (2015).</li></ol>

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...

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).

<table>
	<tbody>
		<tr>
			<td>A new generation of full touch screen gradient PCR instrument</td>
			<td>LongGene</td>
			<td>A200</td>
			<td>Target gene amplification</td>
		</tr>
		<tr>
			<td>AscI restriction enzymes</td>
			<td>New England Biolabs</td>
			<td>R0558V</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>BbsI restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>Clean workbench</td>
			<td>AIRTECH</td>
			<td>SW-CJ-2FD/VS-1300L-U</td>
			<td>A partial purification device in the form of a vertical laminar flow, which creates a local high clean air environment</td>
		</tr>
		<tr>
			<td>DH5&#945; Competent Cells</td>
			<td>TaKaRa</td>
			<td>K613</td>
			<td>Plasmid vector transformation</td>
		</tr>
		<tr>
			<td>Dual-Luciferas Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>Dual-luciferas reporter assay</td>
		</tr>
		<tr>
			<td>Electric thermostatic water bath</td>
			<td>Sanfa Scientific Instruments</td>
			<td>DK-S24</td>
			<td>Heating reagent by constant temperature in water bath</td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td>Beijing Liuyi Biotechnology Co., Ltd.</td>
			<td>DYY-6C</td>
			<td>Control voltage, current, etc.</td>
		</tr>
		<tr>
			<td>Eppendorf Reference 2</td>
			<td>Eppendorf China Ltd.</td>
			<td>Reference 2</td>
			<td>Accurately draw and transfer traces of liquid</td>
		</tr>
		<tr>
			<td>Gel imaging analyzer</td>
			<td>Beijing Liuyi Biotechnology Co., Ltd.</td>
			<td>WD-9413B</td>
			<td>For the analysis of electrophoresis gel images</td>
		</tr>
		<tr>
			<td>GloMax 20/20 Luminometer</td>
			<td>Promega</td>
			<td>E5311</td>
			<td>Detect dual luciferase activity</td>
		</tr>
		<tr>
			<td>High speed refrigerated centrifuge</td>
			<td>BMH</td>
			<td>sigma 3K15</td>
			<td>Nucleic acid extraction and purification</td>
		</tr>
		<tr>
			<td>Intelligent biochemical incubator</td>
			<td>Sanfa Scientific Instruments</td>
			<td>SHP-160</td>
			<td>Provide a suitable temperature environment for the enzyme digestion experiment</td>
		</tr>
		<tr>
			<td>LB Broth Agar</td>
			<td>Sangon Biotech</td>
			<td>A507003-0250</td>
			<td>For the cultivation of E.coli</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent Kit</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td>DNA Transfection</td>
		</tr>
		<tr>
			<td>SalI restriction enzymes</td>
			<td>New England Biolabs</td>
			<td>R3138V</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>SanPrep Column DNA Gel Extraction Kit</td>
			<td>Sangon Biotech</td>
			<td>B518131-0050</td>
			<td>Recycling DNA fragments</td>
		</tr>
		<tr>
			<td>SanPrep Column Plasmid Mini-Preps Kit</td>
			<td>Sangon Biotech</td>
			<td>B518191-0100</td>
			<td>Extraction of plasmid DNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202V</td>
			<td>Link DNA fragment</td>
		</tr>
		<tr>
			<td>TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0</td>
			<td>TaKaRa</td>
			<td>9761</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>Vertical pressure steam sterilizer</td>
			<td>JIBIMED</td>
			<td>LS-50LD</td>
			<td>High temperature and autoclave to kill bacteria, fungi and other microorganisms in laboratory equipment</td>
		</tr>
		<tr>
			<td>Water bath thermostat</td>
			<td>Changzhou Guoyu Instrument Manufacturing Co., Ltd.</td>
			<td>SHZ-82</td>
			<td>Let the bacteria keep shaking, which is good for contact with air.</td>
		</tr>
	</tbody>
</table>

construction of crispr plasmids and detection of knockout efficiency in mammalian cells through a dual luciferase reporter system

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).

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...

Watch this Scientific Journal Video about Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System at JoVE.com

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

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.

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 protocol describes the key steps in the generation and optimization of efficient sgRNA vectors. Our preacceleration of candidate sgRNA targets relative time and inference. The main advantage of this protocol is that it makes it possible to analyze the DNA coding impressions for each of the sgRNAs without editing endogenous genes.This pre-selection method can also be applied to other gene editing measures through double-stranded breaks. Begin by diluting the lyophilized oligonucleotides to a final concentration of 10 micromolar in double distilled water. Mix forward and reverse oligonucleotides in a thin-walled PCR tube at a one-to-one ratio without adding any extra buffer.Incubate the mixture at 95 degrees Celsius for five minutes, then ramp down the temperature to 72 degrees Celsius for 10 minutes. Remove the oligonucleotide mix from the PCR machine and allow it to cool at room temperature. To digest the pX330-xCas9 vector, with Bbs1, combine the vector, enzyme, and digestion buffer in a 50 microliter volume and incubate the reaction at 37 degrees Celsius for two hours.After performing cell transformation, choose five to 10 bacterial colonies from the LB plate and use each of them to inoculate one milliliter of LB media with 60 milligrams ampicillin in a 1.5 milliliter tube. Then incubate the tubes on a rotary shaker for two to three hours. Perform PCR to screen for recombinant plasmids as described in the text manuscript, then run the products on a 2%Agarose gel under 10 volts per centimeter.After identifying the positive plasmids, use them, along with a reporter vector, to co-transfect an appropriate cell line. To lyse the cells, remove the growth medium from the cultured cells. Gently wash the surface of the culture vessel with PBS and dispense 100 microliters of PLB into each culture well to completely cover the cell mono layer.Let the plate incubate at room temperature for 20 minutes, then transfer the lysate to a tube or vial for further handling or storage. To perform the dual luciferase assay, program the luminometers to provide a two second pre-read delay followed by a 10 second measurement period. Then, dispense 100 microliters of luciferase assay reagent into the appropriate number of luminometer tubes.Carefully add 20 microliters of cell lysate into the luminometer tube and mix by pipetting two or three times. Place the tube in the luminometer and initiate the reading. Remove the sample tube from the luminometer.Add 100 microliters of stop reagent and vortex briefly to mix. Return the sample to the luminometer and initiate reading. Record the Renilla Luciferase activity, normalized to the Firefly Luciferase activity, specifically the reciprocal of the ratio displayed on screen.Discard the reaction tube when finished. This method was used to generate three sgRNA vectors for sheep DKK2 Exon 1. sgRNA and xCas9 expressing vectors were built by pre-digesting the vector backbone followed by ligating it in a series of short, double-strand DNA fragments through annealing oligo pairs.Seven bacterial colonies were screened with specific primer pair guided PCR and the positive colonies were detected. The gene targeting capacities of pX330-xCas9 T1, T2, and T3 were simultaneously detected with the dual luciferase assay. The last sgRNA vector displayed the highest detection signal and was subsequently used for sheep gene editing research.Following this protocol, a T7EI assay or a Ribonuclease assay can be performed. These additional measures give more accurate impressions of endogenous genes editing.

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Research

JoVE Journal

Bioengineering

6.1K vues.  Qingdao Agricultural University, Qingdao, China. Ce protocole décrit les étapes clés de la génération et de l’optimisation des vecteurs sgRNA efficaces. Notre préacceleration du candidat sgRNA cible le temps relatif et l’inférence. Le principal avantage de ce protocole est qu’il permet d’analyser les impressions de codage de l’ADN pour chacun des SGARN sans modifier les gènes endogènes.Cette méthode de présélection peut également être appliquée à d’autres mesures d’édition génétique par des ruptures ?...