3.1 Target Selection
for gRNA(s)
3.1.1 Getting to Know
Your Gene(s) of Interest
Sequence, expression and phenotypic information on your gene of
interest should be obtained before initiating gRNA(s) target
selection.
1. Search for your gene of interest on the Sol Genomics Network
to retrieve the genomic sequence with annotated exon-intron
structure, any alternative splicing variants (see Note 8) and the
amino acid sequence.
2. Search for your gene of interest in the Tomato 360 variants
SL.50 data set in JBrowse and select the track of your
S. lycopersicum cultivar of interest (see Note 9) to check if
there are any single nucleotide polymorphisms (SNPs) present
in the protein coding sequence compared to the reference
sequence.
3. Search the literature for your gene of interest. In particular,
look for functional characterization, functionally important
protein domains and redundant genes.
4. Identify paralogous genes using Sol Genomics Network
BLAST and compare their gene expression pattern with that
of your gene of interest on TomExpress to identify potentially
redundant genes (see Note 10).
5. Predict protein domains important for your gene of interest’s
function using InterPro.
3.1.2 Target Region
Selection for gRNA(s)
Select a target region (see Note 11) in the genomic sequence of
your gene of interest using the information gathered in Subheading
3.1.1 to take the following considerations into account.
1. The target region should consist of an early (see Note 12) and
constitutive (see Note 13) protein-coding exon.
2. The target region should be located downstream of (potential)
alternative first exons and in-frame start codons (see Note 14).
3. The target region should be located upstream of regions coding for (predicted) functionally important protein domains (see
Note 15).
3.1.3 Target Site
Selection for gRNA(s)
Select a target site, called a protospacer, immediately preceding a
PAM (see Note 16), within the target region selected in Subheading 3.1.2 and using the information gathered in Subheading 3.1.1.
1. Enter the sequence of your selected target region (see Note 17)
as input for CRISPOR and select the latest S. lycopersicum
genome version and PAM requirement for your chosen Cas
nuclease from the drop-down menus.
2. Select a protospacer of 17–20 nucleotides for one or two
gRNAs (see Notes 18 and 19) that (a) has good on- and
CRISPR-Cas-Mediated Gene Knockout
329
for gRNA(s)
3.1.1 Getting to Know
Your Gene(s) of Interest
Sequence, expression and phenotypic information on your gene of
interest should be obtained before initiating gRNA(s) target
selection.
1. Search for your gene of interest on the Sol Genomics Network
to retrieve the genomic sequence with annotated exon-intron
structure, any alternative splicing variants (see Note 8) and the
amino acid sequence.
2. Search for your gene of interest in the Tomato 360 variants
SL.50 data set in JBrowse and select the track of your
S. lycopersicum cultivar of interest (see Note 9) to check if
there are any single nucleotide polymorphisms (SNPs) present
in the protein coding sequence compared to the reference
sequence.
3. Search the literature for your gene of interest. In particular,
look for functional characterization, functionally important
protein domains and redundant genes.
4. Identify paralogous genes using Sol Genomics Network
BLAST and compare their gene expression pattern with that
of your gene of interest on TomExpress to identify potentially
redundant genes (see Note 10).
5. Predict protein domains important for your gene of interest’s
function using InterPro.
3.1.2 Target Region
Selection for gRNA(s)
Select a target region (see Note 11) in the genomic sequence of
your gene of interest using the information gathered in Subheading
3.1.1 to take the following considerations into account.
1. The target region should consist of an early (see Note 12) and
constitutive (see Note 13) protein-coding exon.
2. The target region should be located downstream of (potential)
alternative first exons and in-frame start codons (see Note 14).
3. The target region should be located upstream of regions coding for (predicted) functionally important protein domains (see
Note 15).
3.1.3 Target Site
Selection for gRNA(s)
Select a target site, called a protospacer, immediately preceding a
PAM (see Note 16), within the target region selected in Subheading 3.1.2 and using the information gathered in Subheading 3.1.1.
1. Enter the sequence of your selected target region (see Note 17)
as input for CRISPOR and select the latest S. lycopersicum
genome version and PAM requirement for your chosen Cas
nuclease from the drop-down menus.
2. Select a protospacer of 17–20 nucleotides for one or two
gRNAs (see Notes 18 and 19) that (a) has good on- and
CRISPR-Cas-Mediated Gene Knockout
329