5. Analyze the predicted gRNA secondary structure by comparing it to its optimal structure (Fig. 1) (see Subheading 1.5) (see
Note 9).
6. Guide(s) of suitable gRNAs can subsequently be used for cloning of the CRISPR constructs and experimental procedure.
4 Notes
1. The optimal guide length depends on the employed CRISPR/
Cas-system, respective Cas ortholog, as well as experimental
system. The most commonly used SpCas9 and SaCas9 show
solid efficiency among the majority of experimental systems
with a 20-nt guide.
2. Cloning of the guide is commonly realized by its synthesis as
oligonucleotides with subsequent oligonucleotide annealing
and ligation into the linearized gRNA expression vector. The
addition of 5
0 -overhangs to the oligonucleotides enables stickyend cloning which guarantees integration of the guide in the
correct orientation.
3. In general, a total amount of 4 mismatches between guide
and target site or 2 mismatches between guide and target site
within the first seven nucleotides proximal to the PAM is
sufficient to prevent the majority of cleavage activity. Thus, if
the amount of total mismatches is 4, at least two mismatches
should be inside this so-called core region to prevent off-target
activity. The “core” region is defined as the region being the
most sensitive toward mismatches.
4. A high efficacy score is desirable. If off-target sites exist, 4
mismatches in total or 2 mismatches in the “core” region
prevent the majority of cleavage. Select the target(s) that have
the highest efficacy score while showing the lowest likelihood
for off-target activity.
5. This option is only available for the Cas-Designer tool and
consequently complicates the comparison between the
CCTop and Cas-Designer results in this step.
6. The “out-of-frame score” describes the likelihood for the
emergence of out-of-frame mutations caused by the
microhomology-mediated end-joining pathway. Out-of-frame
mutations are desired for knockout experiments.
7. Targets with two mismatches within the “core” region can still
be selected.
8. Prevent selecting targets that show potential off-target sites by
only having one 1 nt DNA bulge in total or one 1 nt RNA
bulge outside the “core” region. One 2 nt DNA bulge in total
Guidelines for gRNA Design
339
Note 9).
6. Guide(s) of suitable gRNAs can subsequently be used for cloning of the CRISPR constructs and experimental procedure.
4 Notes
1. The optimal guide length depends on the employed CRISPR/
Cas-system, respective Cas ortholog, as well as experimental
system. The most commonly used SpCas9 and SaCas9 show
solid efficiency among the majority of experimental systems
with a 20-nt guide.
2. Cloning of the guide is commonly realized by its synthesis as
oligonucleotides with subsequent oligonucleotide annealing
and ligation into the linearized gRNA expression vector. The
addition of 5
0 -overhangs to the oligonucleotides enables stickyend cloning which guarantees integration of the guide in the
correct orientation.
3. In general, a total amount of 4 mismatches between guide
and target site or 2 mismatches between guide and target site
within the first seven nucleotides proximal to the PAM is
sufficient to prevent the majority of cleavage activity. Thus, if
the amount of total mismatches is 4, at least two mismatches
should be inside this so-called core region to prevent off-target
activity. The “core” region is defined as the region being the
most sensitive toward mismatches.
4. A high efficacy score is desirable. If off-target sites exist, 4
mismatches in total or 2 mismatches in the “core” region
prevent the majority of cleavage. Select the target(s) that have
the highest efficacy score while showing the lowest likelihood
for off-target activity.
5. This option is only available for the Cas-Designer tool and
consequently complicates the comparison between the
CCTop and Cas-Designer results in this step.
6. The “out-of-frame score” describes the likelihood for the
emergence of out-of-frame mutations caused by the
microhomology-mediated end-joining pathway. Out-of-frame
mutations are desired for knockout experiments.
7. Targets with two mismatches within the “core” region can still
be selected.
8. Prevent selecting targets that show potential off-target sites by
only having one 1 nt DNA bulge in total or one 1 nt RNA
bulge outside the “core” region. One 2 nt DNA bulge in total
Guidelines for gRNA Design
339