Paste selected sequence in the selected online tool.
Parameters that should be considered are:
\n
Consider choosing several different target sites, since not all sgRNAs work equally well.
Consider using a 20 bp target site that also contains a recognition site of a commercially available restriction enzyme. The target site of the restriction enzyme should span the site of Cas9 cleavage (3 bp in front of the PAM). This will allow you to use the restriction enzyme length polymorphism assay to detect mutations in your gene of interest.
Genomic DNA region must correspond to the crRNA sequence of the sgRNA:
The 3' end of the DNA target sequence must have a PAM sequence. The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA), and Cas9 nuclease will cleave approximately three bases upstream of the PAM.
Verify eight E. coli colonies by colony PCR using primer and your protospacer primer with the following setup.
PCR program:
Transform competent E. coli cells with the ligation reaction and spread the transformed cells on LB agar plates supplemented with Amp.
Recipe:
15 g/L Bacto-tryptone
5 g/L yeast extract
5 g/L NaCl
with 200 μg/mL ampicillin
Digesting plasmid with a restriction enzyme for 3 hours at 37°C. Run it on an agarose gel and extract the vector backbone from the gel using the DNA Purification Kit.
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For example:
Forward primer: TTCG-GN19
Reverse primer: AAAC-N19
Pipette 10 μL of each primer dilution into a 1.5 mL microcentrifuge tube and mix.
Heat primer mix:
Transform competent E. coli cells with the reaction mixture and spread the transformed cells on LB agar plates supplemented with Kan.
LB-medium:
Amplify it from a plasmid with target sequence using primers and polymerase and then extract the sgRNA cassette from the gel. Assemble the plasmid backbone and the sgRNA cassette to generate the final vector using Assembly Master Mix according to the manufacturer’s instruction with an incubation time of 1 h.
PCR program:
Inoculate positive colony into LB medium supplemented with Kan and let grow overnight.
LB-medium:
Digestion of a plasmid with restriction enzymes.
Conditions:
Verify eight positive colonies by colony PCR.
PCR program:
With 1 μg of (pUB-Cas9-gene of interest) and spread the transformed cells on YEP agar plates supplemented with Rif/Gent/Kan.
Recipe:
Verify plasmid presence in at least three colonies by colony PCR.
\nTransform plants using Agrobacterium-mediated T-DNA transfer with the floral dipping method.
Simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L‐77. Sucrose and surfactant are critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques are present produce transformed progeny at the highest rate.
PCR/RE Genotyping: Genotyping of clones with targeted mutation
DNA sequencing
The transgene free homozygous mutants with desired genetic modifications at the targeted loci could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.
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