37. Colony-PCR and Sanger sequencing are not encouraged here
because of the tandem array of gRNAs produced.
38. Fragment size after digestion will vary depending on the binary
vector used in step 1.
39. As hairy root cultures obtained by rhizogenic Agrobacteriummediated transformation of CRISPR-Cas are often chimeric,
make sure to cut off approximately 1.0 cm from the distal end
of a single, lateral root that was recently subcultured. This will
ensure that the Sanger sequencing data obtained in Subheading 3.5.3 will be representative for that recent hairy root
subculture.
40. In case leaf material needs to be collected from primary transformant (T0) plant lines, only collect it from well-growing
regenerated explants.
41. Use gDNA from a wild-type plant as a negative control template and the used binary vector as a positive control template.
42. Use gDNA from a wild-type plant as a control template and
Milli-Q water as a no-template control.
43. Use PCR-tubes (eight-strip format).
44. The beads will be pulled to the magnet.
45. Keep samples on the magnetic separator until step 12.
46. Do this without disturbing the beads.
47. All traces of ethanol need to be removed before proceeding to
the next step, but beware not to over dry the beads to avoid
cracking and subsequent yield loss.
48. Sequence with the PCR primer located 150–250 bp from the
Cas-cleavage site used in step 1 of Subheading 3.5.3.
49. Indels that are not a multiple of three bases will shift the open
reading frame, which can result in the translation of a truncated
nonfunctional protein and/or nonsense-mediated decay triggered by a premature stop codon [11].
50. Alternatively, CRISPR/Cas-induced mutations can be analyzed using cleaved amplified polymorphic sequence (CAPS),
T7 endonuclease, next-generation sequencing and highresolution melting curve analysis [12]. However, Sanger
sequencing followed by ICE analysis is the most cost-effective
option.
51. Use the 2C-peak of nuclei isolated from a wild-type leaf (diploid) for calibration.
52. Likewise, a primary transformant (T0) plant line that has difficulty setting seed can be rescued by crossing it to the wild-type.
53. A flower that is still closed is chosen, because it is unlikely to
have self-pollinated.
CRISPR-Cas-Mediated Gene Knockout
339
because of the tandem array of gRNAs produced.
38. Fragment size after digestion will vary depending on the binary
vector used in step 1.
39. As hairy root cultures obtained by rhizogenic Agrobacteriummediated transformation of CRISPR-Cas are often chimeric,
make sure to cut off approximately 1.0 cm from the distal end
of a single, lateral root that was recently subcultured. This will
ensure that the Sanger sequencing data obtained in Subheading 3.5.3 will be representative for that recent hairy root
subculture.
40. In case leaf material needs to be collected from primary transformant (T0) plant lines, only collect it from well-growing
regenerated explants.
41. Use gDNA from a wild-type plant as a negative control template and the used binary vector as a positive control template.
42. Use gDNA from a wild-type plant as a control template and
Milli-Q water as a no-template control.
43. Use PCR-tubes (eight-strip format).
44. The beads will be pulled to the magnet.
45. Keep samples on the magnetic separator until step 12.
46. Do this without disturbing the beads.
47. All traces of ethanol need to be removed before proceeding to
the next step, but beware not to over dry the beads to avoid
cracking and subsequent yield loss.
48. Sequence with the PCR primer located 150–250 bp from the
Cas-cleavage site used in step 1 of Subheading 3.5.3.
49. Indels that are not a multiple of three bases will shift the open
reading frame, which can result in the translation of a truncated
nonfunctional protein and/or nonsense-mediated decay triggered by a premature stop codon [11].
50. Alternatively, CRISPR/Cas-induced mutations can be analyzed using cleaved amplified polymorphic sequence (CAPS),
T7 endonuclease, next-generation sequencing and highresolution melting curve analysis [12]. However, Sanger
sequencing followed by ICE analysis is the most cost-effective
option.
51. Use the 2C-peak of nuclei isolated from a wild-type leaf (diploid) for calibration.
52. Likewise, a primary transformant (T0) plant line that has difficulty setting seed can be rescued by crossing it to the wild-type.
53. A flower that is still closed is chosen, because it is unlikely to
have self-pollinated.
CRISPR-Cas-Mediated Gene Knockout
339