7. Although magnetic beads for PCR amplicon purification may
be purchased from any supplier, steps 2–19 in Subheading
3.5.3 apply specifically to the use of HighPrep™ PCR reagent
from MAGBIO.
8. In the imaginary Solyc identifier Solyc01g001010.1.1, the
second “.1” denotes the splice variant.
9. Look up the accession number of your cultivar of interest in the
Tomato Genetics Resource Center Accessions database.
10. Paralogous genes with overlapping expression patterns might
code for proteins with redundant functions.
11. If (potentially) redundant genes were identified in steps 3
and/or 4 of Subheading 3.1.1, targeting a conserved region
might be considered.
12. Mutagenesis in the 3
0 end coding region or introns may not
result in null mutations.
13. Do not target exons that are excluded in alternative splice
variants to ensure complete gene function disruption, unless
you want to impair the function of a protein encoded by a
particular splice variant only.
14. N-terminal protein truncations, resulting from mutagenesis in
a region upstream of an alternative first exon or in-frame start
codon, may have little or no effect on gene function.
15. Mutagenesis downstream of functionally important protein
domains may not lead to completely impaired protein function.
16. If (potentially) redundant genes were identified in step 3
and/or 4 of Subheading 3.1.1, targeting a conserved site
might be considered.
17. Adapt the sequence of your selected target region according to
any SNPs that may be present in your S. lycopersicum cultivar of
choice (see step 2 of Subheading 3.1.1).
18. Using two gRNAs to target your gene of interest can cause a
bigger genomic deletion, ensuring the interruption of gene
function.
19. When selecting two target sites (for two gRNAs), the
Cas-cleavage sites should be out of frame in the coding
sequence. The cleavage site is specific for each Cas nuclease
and is located 3–4 bp upstream of the PAM sequence for Cas9.
20. Mismatches of the protospacer within the target region distal
to the PAM are generally tolerated, whereas complete complementarity with the 8–12 bp immediately preceding the PAM is
crucial for efficient cleavage by the Cas nuclease.
21. In case there are no protospacers available with both good onand off-target scores, consider what is most important for your
project.
CRISPR-Cas-Mediated Gene Knockout
337
be purchased from any supplier, steps 2–19 in Subheading
3.5.3 apply specifically to the use of HighPrep™ PCR reagent
from MAGBIO.
8. In the imaginary Solyc identifier Solyc01g001010.1.1, the
second “.1” denotes the splice variant.
9. Look up the accession number of your cultivar of interest in the
Tomato Genetics Resource Center Accessions database.
10. Paralogous genes with overlapping expression patterns might
code for proteins with redundant functions.
11. If (potentially) redundant genes were identified in steps 3
and/or 4 of Subheading 3.1.1, targeting a conserved region
might be considered.
12. Mutagenesis in the 3
0 end coding region or introns may not
result in null mutations.
13. Do not target exons that are excluded in alternative splice
variants to ensure complete gene function disruption, unless
you want to impair the function of a protein encoded by a
particular splice variant only.
14. N-terminal protein truncations, resulting from mutagenesis in
a region upstream of an alternative first exon or in-frame start
codon, may have little or no effect on gene function.
15. Mutagenesis downstream of functionally important protein
domains may not lead to completely impaired protein function.
16. If (potentially) redundant genes were identified in step 3
and/or 4 of Subheading 3.1.1, targeting a conserved site
might be considered.
17. Adapt the sequence of your selected target region according to
any SNPs that may be present in your S. lycopersicum cultivar of
choice (see step 2 of Subheading 3.1.1).
18. Using two gRNAs to target your gene of interest can cause a
bigger genomic deletion, ensuring the interruption of gene
function.
19. When selecting two target sites (for two gRNAs), the
Cas-cleavage sites should be out of frame in the coding
sequence. The cleavage site is specific for each Cas nuclease
and is located 3–4 bp upstream of the PAM sequence for Cas9.
20. Mismatches of the protospacer within the target region distal
to the PAM are generally tolerated, whereas complete complementarity with the 8–12 bp immediately preceding the PAM is
crucial for efficient cleavage by the Cas nuclease.
21. In case there are no protospacers available with both good onand off-target scores, consider what is most important for your
project.
CRISPR-Cas-Mediated Gene Knockout
337