1. Mix 100 ng of each of the six Golden Gate entry modules with
100 ng of binary vector (see Note 36), 5 units of BsaI-HFv2,
200 units of T4 DNA ligase, 1.5 μL of 10 mM ATP, 1.5 μL of
10Â recommended restriction enzyme buffer, and 9 μL of
Milli-Q water in a PCR tube.
2. Place in a thermal cycler and run the following program to
perform a Golden Gate reaction: (37
C for 3 min, 16
C for
3 min) Â 30 cycles, 50
C for 5 min, 80
C for 5 min, and hold
at 12
C.
3. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with 75 μg/mL
spectinomycin.
4. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
5. Perform a diagnostic digest with NheI on ~1 μg of binary
vector to verify the presence of your gRNA(s) and linker if
applicable (see Notes 37 and 38).
3.4 Vector Delivery
The two predominant stable tomato transformation systems used
to obtain gene knockouts using CRISPR-Cas are rhizogenic Agrobacterium- and Agrobacterium tumefaciens-mediated transformation (see Table 1). Rhizogenic Agrobacterium-induced hairy root
cultures edited by CRISPR-Cas can be obtained within 2 months
for rapid functional characterization of genes expressed in the root
of S. lycopersicum [3]. Although generating CRISPR-Cas-mediated
gene knockout plant lines using A. tumefaciens is more laborintensive and time-consuming because of lower transformation
efficiencies and long generation times [4–6], it allows for the
investigation of gene function in any tissue and developmental
stage.
1. Transform your binary vector into rhizogenic Agrobacterium
or A. tumefaciens.
2. Stably transform S. lycopersicum via rhizogenic Agrobacteriumor A. tumefaciens-mediated transformation (see Table 1).
3.5 Identification
of Gene Knockout
Lines
In this section, we describe how to identify stable gene knockout
lines obtained through rhizogenic Agrobacterium- or
A. tumefaciens-mediated transformation of CRISPR-Cas vectors.
3.5.1 Rapid gDNA
Extraction
1. Collect approximately 1.0 cm of hairy root material (see Note
39) or 1.0 cm
2 of leaf material (see Note 40) in microtiter tubes
(eight-strip format) filled with two stainless steel beads (diameter 3.0 mm).
2. Dip in liquid nitrogen for flash freezing or store at À80
C for
at least 1 h.
CRISPR-Cas-Mediated Gene Knockout
333
100 ng of binary vector (see Note 36), 5 units of BsaI-HFv2,
200 units of T4 DNA ligase, 1.5 μL of 10 mM ATP, 1.5 μL of
10Â recommended restriction enzyme buffer, and 9 μL of
Milli-Q water in a PCR tube.
2. Place in a thermal cycler and run the following program to
perform a Golden Gate reaction: (37
C for 3 min, 16
C for
3 min) Â 30 cycles, 50
C for 5 min, 80
C for 5 min, and hold
at 12
C.
3. Transform 5 μL of the Golden Gate reaction into competent
E. coli DH5α and plate on LB medium with 75 μg/mL
spectinomycin.
4. Isolate binary vector DNA from two colonies using a plasmid
miniprep kit.
5. Perform a diagnostic digest with NheI on ~1 μg of binary
vector to verify the presence of your gRNA(s) and linker if
applicable (see Notes 37 and 38).
3.4 Vector Delivery
The two predominant stable tomato transformation systems used
to obtain gene knockouts using CRISPR-Cas are rhizogenic Agrobacterium- and Agrobacterium tumefaciens-mediated transformation (see Table 1). Rhizogenic Agrobacterium-induced hairy root
cultures edited by CRISPR-Cas can be obtained within 2 months
for rapid functional characterization of genes expressed in the root
of S. lycopersicum [3]. Although generating CRISPR-Cas-mediated
gene knockout plant lines using A. tumefaciens is more laborintensive and time-consuming because of lower transformation
efficiencies and long generation times [4–6], it allows for the
investigation of gene function in any tissue and developmental
stage.
1. Transform your binary vector into rhizogenic Agrobacterium
or A. tumefaciens.
2. Stably transform S. lycopersicum via rhizogenic Agrobacteriumor A. tumefaciens-mediated transformation (see Table 1).
3.5 Identification
of Gene Knockout
Lines
In this section, we describe how to identify stable gene knockout
lines obtained through rhizogenic Agrobacterium- or
A. tumefaciens-mediated transformation of CRISPR-Cas vectors.
3.5.1 Rapid gDNA
Extraction
1. Collect approximately 1.0 cm of hairy root material (see Note
39) or 1.0 cm
2 of leaf material (see Note 40) in microtiter tubes
(eight-strip format) filled with two stainless steel beads (diameter 3.0 mm).
2. Dip in liquid nitrogen for flash freezing or store at À80
C for
at least 1 h.
CRISPR-Cas-Mediated Gene Knockout
333