2. Enter your gene/sequence of interest into the query sequence
field. For knockout experiments, concentrate on the exons of
the gene (see Subheading 1.8).
3. Select the PAM type. The PAM depends on the employed
CRISPR/Cas-system and respective Cas ortholog (e.g.,
5
0 -NGG-3
0 for Streptococcus pyogenes Cas9).
4. Select the guide length of the respective CRISPR ortholog.
This sequence corresponds to the nucleotides upstream of the
PAM for Cas9 (see Note 1).
5. Optional: Enter 5
0 and 3
0 target site limitations. Dependent on
the promoter and/or experimental system, the occurrence of
specific nucleotides at these positions increases editing efficiency (see Subheading 1.3).
6. Optional: Specify the 5
0 guide sequence overhangs. Cloning of
the guide is commonly realized via oligonucleotide annealing
and sticky-end ligation (see Note 2).
7. For off-target prediction, select the number of total mismatches between guide and target site to be considered. Recommendation: Select four (see Subheading 1.4) (see Note 3).
8. Optional: Define the core length of the respective CRISPR
ortholog and the number of total core mismatches between
guide and target site to be considered. Recommendation:
seven for core length and two for core mismatches (see Subheading 1.4) (see Note 3).
9. Select the target genome and submit the request.
10. Choose the guide(s) with the best efficacy score/off-target
ratio (see Note 4). Compare the results with Cas-Designer to
identify the optimal guide(s).
3.1.2 CRISPR RGEN
Tools: Cas-Designer
1. Go to http://www.rgenome.net/cas-designer/ to open the
Cas-Designer tool.
2. Select the PAM type. The PAM depends on the employed
CRISPR/Cas-system and respective Cas ortholog (e.g.,
5
0 -NGG-3
0 for Streptococcus pyogenes Cas9).
3. Select the target genome.
4. Enter your target gene/sequence of interest into the query
sequence field. For knockout experiments, concentrate on the
exons of the gene (see Subheading 1.8).
5. Select the guide length (here: crRNA length) of the respective
CRISPR ortholog (see Note 1).
6. Optional: Allow integration of 1 nt bulge for off-target analysis
(see Subheading 1.4). However, this is not recommended in
this step (see Note 5).
7. Submit the request.
Guidelines for gRNA Design
337
field. For knockout experiments, concentrate on the exons of
the gene (see Subheading 1.8).
3. Select the PAM type. The PAM depends on the employed
CRISPR/Cas-system and respective Cas ortholog (e.g.,
5
0 -NGG-3
0 for Streptococcus pyogenes Cas9).
4. Select the guide length of the respective CRISPR ortholog.
This sequence corresponds to the nucleotides upstream of the
PAM for Cas9 (see Note 1).
5. Optional: Enter 5
0 and 3
0 target site limitations. Dependent on
the promoter and/or experimental system, the occurrence of
specific nucleotides at these positions increases editing efficiency (see Subheading 1.3).
6. Optional: Specify the 5
0 guide sequence overhangs. Cloning of
the guide is commonly realized via oligonucleotide annealing
and sticky-end ligation (see Note 2).
7. For off-target prediction, select the number of total mismatches between guide and target site to be considered. Recommendation: Select four (see Subheading 1.4) (see Note 3).
8. Optional: Define the core length of the respective CRISPR
ortholog and the number of total core mismatches between
guide and target site to be considered. Recommendation:
seven for core length and two for core mismatches (see Subheading 1.4) (see Note 3).
9. Select the target genome and submit the request.
10. Choose the guide(s) with the best efficacy score/off-target
ratio (see Note 4). Compare the results with Cas-Designer to
identify the optimal guide(s).
3.1.2 CRISPR RGEN
Tools: Cas-Designer
1. Go to http://www.rgenome.net/cas-designer/ to open the
Cas-Designer tool.
2. Select the PAM type. The PAM depends on the employed
CRISPR/Cas-system and respective Cas ortholog (e.g.,
5
0 -NGG-3
0 for Streptococcus pyogenes Cas9).
3. Select the target genome.
4. Enter your target gene/sequence of interest into the query
sequence field. For knockout experiments, concentrate on the
exons of the gene (see Subheading 1.8).
5. Select the guide length (here: crRNA length) of the respective
CRISPR ortholog (see Note 1).
6. Optional: Allow integration of 1 nt bulge for off-target analysis
(see Subheading 1.4). However, this is not recommended in
this step (see Note 5).
7. Submit the request.
Guidelines for gRNA Design
337