3 Methods
3.1 Single/Multiple
Gene Mutations and
Base Editing
3.1.1 Design of sgRNA
sgRNAs design is crucial to achieve the specificity and efficiency of
the CRISPR/Cas9 system. Nowadays, a number of web-based
tools are available that can help with quick sgRNA design. These
tools are very useful for predicting the efficiency of sgRNAs and
potential off-target sites. sgRNA design tools described below
generate a list of candidate sgRNAs from the input sequence (see
Note 1):
1. CRISPR Primer Designer: http://plantsignal.cn/CRISPR/
crispr_primer_designer.html
2. CRISPR-PLANT: https://www.genome.arizona.edu/crispr/
index.html
3. CRISPOR: http://crispor.tefor.net/
4. CHOPCHOP: http://chopchop.cbu.uib.no/
5. DESKGEN: https://www.deskgen.com/landing/cloud.html
6. CRISPR tool ATUM: https://www.atum.bio/eCommerce/
cas9/input
Alternatively, sgRNAs can be designed manually by identifying
the 20-bp sequence directly upstream of any 5
0 -NGG. A pair of
DNA oligos are synthesized as follows (Fig. 3):
Forward oligo: 5
0 -gattGNNNNNNNNNNNNNNNNNNNN-3
0
Reverse oligo: 5
0 -aaacNNNNNNNNNNNNNNNNNNNNC-3
0
The “N”s in the forward oligo correspond to the 5
0 20-nt
preceding the PAM (5
0 -NGG-3
0 ) and those in the reverse oligo
are just the reverse complementary sequence (Fig. 3c) (see Note 2).
To target multiple genes simultaneously, a single or multiple
sgRNAs can be designed. When target genes share a conserved
DNA sequence, designing an sgRNA from this sequence will be
enough to generate multiple gene mutations, otherwise genespecific multiple sgRNAs are designed (Fig. 2).
3.1.2 sgRNA Order and
Dilution
Order the designed oligomers for sgRNA. Dissolve and dilute the
sgRNA oligomers in ddH 2 O to get a final concentration of 100 μM
and store them at À20
C.
3.1.3 Preparation of
sgRNA Construct
1. Anneal each pair of DNA oligomers (Fig. 3a, c):
1 μL Forward oligo (100 μM)
1 μL Reverse oligo (100 μM)
Add ddH 2 O to 10 μL total reaction volume.
CRISPR/Cas9-Based Genome Editing in Arabidopsis
131
3.1 Single/Multiple
Gene Mutations and
Base Editing
3.1.1 Design of sgRNA
sgRNAs design is crucial to achieve the specificity and efficiency of
the CRISPR/Cas9 system. Nowadays, a number of web-based
tools are available that can help with quick sgRNA design. These
tools are very useful for predicting the efficiency of sgRNAs and
potential off-target sites. sgRNA design tools described below
generate a list of candidate sgRNAs from the input sequence (see
Note 1):
1. CRISPR Primer Designer: http://plantsignal.cn/CRISPR/
crispr_primer_designer.html
2. CRISPR-PLANT: https://www.genome.arizona.edu/crispr/
index.html
3. CRISPOR: http://crispor.tefor.net/
4. CHOPCHOP: http://chopchop.cbu.uib.no/
5. DESKGEN: https://www.deskgen.com/landing/cloud.html
6. CRISPR tool ATUM: https://www.atum.bio/eCommerce/
cas9/input
Alternatively, sgRNAs can be designed manually by identifying
the 20-bp sequence directly upstream of any 5
0 -NGG. A pair of
DNA oligos are synthesized as follows (Fig. 3):
Forward oligo: 5
0 -gattGNNNNNNNNNNNNNNNNNNNN-3
0
Reverse oligo: 5
0 -aaacNNNNNNNNNNNNNNNNNNNNC-3
0
The “N”s in the forward oligo correspond to the 5
0 20-nt
preceding the PAM (5
0 -NGG-3
0 ) and those in the reverse oligo
are just the reverse complementary sequence (Fig. 3c) (see Note 2).
To target multiple genes simultaneously, a single or multiple
sgRNAs can be designed. When target genes share a conserved
DNA sequence, designing an sgRNA from this sequence will be
enough to generate multiple gene mutations, otherwise genespecific multiple sgRNAs are designed (Fig. 2).
3.1.2 sgRNA Order and
Dilution
Order the designed oligomers for sgRNA. Dissolve and dilute the
sgRNA oligomers in ddH 2 O to get a final concentration of 100 μM
and store them at À20
C.
3.1.3 Preparation of
sgRNA Construct
1. Anneal each pair of DNA oligomers (Fig. 3a, c):
1 μL Forward oligo (100 μM)
1 μL Reverse oligo (100 μM)
Add ddH 2 O to 10 μL total reaction volume.
CRISPR/Cas9-Based Genome Editing in Arabidopsis
131