8. Select a GC content of 25–75% and submit the filter (see
Subheading 1.3).
9. Choose the guide(s) with a “out-of-frame score” above 66 (see
Note 6) and with no potential mismatch targets (see Note 7).
Compare with CCTop results to identify the optimal guide(s).
3.2 Extended
Off-Target Analysis
Online tools for guide sequence selection often include mismatches
only for off-target prediction. However, DNA and RNA bulges also
contribute to off-target activity. Therefore, an extended off-target
analysis using tools including these criteria might be useful to
improve off-target prediction even further.
3.2.1 CRISPR RGEN
Tools: Cas-OFFinder
1. Go to http://www.rgenome.net/cas-offinder/ to open the
Cas-OFFinder 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 the guide sequence(s) into the query sequence field. The
guide sequence(s) equal the guide(s) selected with CCTop/
Cas-Designer.
5. Select the number of total mismatches and the DNA/RNA
bulge size between guide and target site to be considered.
Recommendation: Select three for number of total mismatches, two for DNA bulge, and one for RNA bulge (see
Subheading 1.4) (see Note 8).
6. Submit the request.
7. Choose the guide(s) with no off-target sites or at least high
discrepancy to the predicted off-target sites (see Notes 3
and 8).
3.3 Analysis of gRNA
Secondary Structure
The secondary structure of the gRNA considerably contributes to
overall activity. Being strongly affected by the variable guide
sequence, verification of the secondary structure is inevitable.
3.3.1 RNAfold
1. Go to http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/
RNAfold.cgi to open the RNAfold tool.
2. Enter the complete gRNA sequence including the guide
(s) determined by the previous analysis into the sequence
query field. The guide is upstream of the gRNA backbone.
3. Keep the default settings.
4. Submit the request.
338
Patrick Schindele et al.
Subheading 1.3).
9. Choose the guide(s) with a “out-of-frame score” above 66 (see
Note 6) and with no potential mismatch targets (see Note 7).
Compare with CCTop results to identify the optimal guide(s).
3.2 Extended
Off-Target Analysis
Online tools for guide sequence selection often include mismatches
only for off-target prediction. However, DNA and RNA bulges also
contribute to off-target activity. Therefore, an extended off-target
analysis using tools including these criteria might be useful to
improve off-target prediction even further.
3.2.1 CRISPR RGEN
Tools: Cas-OFFinder
1. Go to http://www.rgenome.net/cas-offinder/ to open the
Cas-OFFinder 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 the guide sequence(s) into the query sequence field. The
guide sequence(s) equal the guide(s) selected with CCTop/
Cas-Designer.
5. Select the number of total mismatches and the DNA/RNA
bulge size between guide and target site to be considered.
Recommendation: Select three for number of total mismatches, two for DNA bulge, and one for RNA bulge (see
Subheading 1.4) (see Note 8).
6. Submit the request.
7. Choose the guide(s) with no off-target sites or at least high
discrepancy to the predicted off-target sites (see Notes 3
and 8).
3.3 Analysis of gRNA
Secondary Structure
The secondary structure of the gRNA considerably contributes to
overall activity. Being strongly affected by the variable guide
sequence, verification of the secondary structure is inevitable.
3.3.1 RNAfold
1. Go to http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/
RNAfold.cgi to open the RNAfold tool.
2. Enter the complete gRNA sequence including the guide
(s) determined by the previous analysis into the sequence
query field. The guide is upstream of the gRNA backbone.
3. Keep the default settings.
4. Submit the request.
338
Patrick Schindele et al.