3. Tomato Genetics Resource Center Accessions (https://tgrc.
ucdavis.edu/Data/Acc/dataframe.aspx?start¼AccSearch.
aspx&navstart¼nav.html).
4. Sol Genomics Network BLAST (https://solgenomics.net/
tools/blast/).
5. TomExpress (http://tomexpress.toulouse.inra.fr/).
6. InterPro (https://www.ebi.ac.uk/interpro/).
7. CRISPOR (http://crispor.tefor.net/).
8. Primer-BLAST
(https://www.ncbi.nlm.nih.gov/tools/
primer-blast/).
9. Inference of CRISPR Editing (ICE) CRISPR Analysis Tool
(https://ice.synthego.com/#/).
10. Software for ploidy level analysis (e.g., FloMax software from
Sysmex).
2.4 Equipment
1. Microtiter tubes (eight-strip format).
2. Stainless steel beads (diameter 3.0 mm).
3. Tissue lyser.
Table 2
Characteristics of MultiSite-Puchta vectors for one or two gRNAs
Vector
Gateway att
sites
gRNA
promoter
Cas
promoter Cas
PAM
E. coli
Marker
Plant
Marker References
Entry
vectors:
pENC1.1 L1-L2
AtU6–26 —
—
Amp
—
[14]
pMR217 L1-R5
AtU6–26 —
—
Km
—
[15, 16]
pMR218 L5-L2
AtU6–26 —
—
Km
—
[15, 16]
Binary
vectors:
Cas9PPT
R1-R2
—
PcUbi4–2 SpCas9
a
NGG Sp
PPT
[14]
Cas9km
R1-R2
—
PcUbi4–2 SpCas9
a
NGG Sp
Km
[15, 16]
Cas9km
R1-R2
—
PcUbi4–2 SpCas9
(VQR)
a
NGA Sp
Km
[17]
Abbreviations: gRNA, guide RNA; PAM, protospacer adjacent motif; Amp, ampicillin; Km, kanamycin; Sp, spectinomycin; PPT, phosphinothricin; Ref., reference
a
Arabidopsis thaliana codon-optimized Streptococcus pyogenes Cas9
CRISPR-Cas-Mediated Gene Knockout
325