abundant protospacer adjacent motif (PAM), apparently providing
a wide variety of potential target sites, and gRNAs. However,
indiscriminately selecting gRNAs can minimize and even prevent
experimental success. Thus, optimization of gRNA design is
required for improving target specificity and maximizing editing
efficiency. Pre-experimental procedures therefore often involve the
screening of different guides to determine the optimal target site.
While animal cell lines allow high-throughput screenings, evaluating gRNA-editing efficiency in other organisms can be an elaborate
process demanding longer periods for the generation of transgenic
individuals. Especially for many crops like maize and wheat, generation of transgenic plants is very time and cost intensive, and being
able to estimate gRNA efficiency before engaging in the laborious
process of transgenic plant production would be most desirable.
For this very reason, guidelines helping to define efficient gRNAs
are of tremendous importance to ensure an optimal experimental
progress. Both target site and gRNA features determine the
on-target and off-target activity of the CRISPR nuclease. Length
and sequence composition or structural features of the guide and
target, respectively, have been reported as main contributors to
overall efficiency.
1.2 Guide Length
Variation for Reduced
Off-Target Activity
The various Cas9 orthologs put different demands on PAM composition and guide length, yet, for each individual ortholog, an
optimal PAM and guide length required for maximum on-target
activity were identified [7]. Based on this observation, the most
commonly used Streptococcus pyogenes Cas9 (SpCas9) is employed
combining a 20-nt guide and the 5
0 -NGG-3
0 PAM. However, due
to the abundance of the PAM and a certain tolerance toward
mismatches, off-target activity was frequently detected in human
and animal cell lines [8, 9]. More recent studies on human 293T
cells and Drosophila reported strongly reduced off-target activity by
employing truncated guides of 17 to 18 nt in length while maintaining the editing efficiency of full-length guides [10–12]. The
strong decrease in off-target activity can presumably be explained
by a much stronger disruptive impact of mismatches on truncated
guides. Unfortunately, contradictory results have been reported in
stem cells and plants where truncated guides were less efficient
than full-length guides [13, 14]. Apart from that, full-length guides
are highly precise in plants and off-target effects only detectable
for highly similar targets or targets with PAM distal mismatches
[15–19].
1.3 Effects
of Nucleotide
Composition
and Identity on Guide
Efficiency
Concerning the effect of the nucleotide composition of the guide
and target, rather inconsistent results have been published. It is
agreed that guides having a very low or very high GC content are
less effective [20–22]. However, analysis about the optimal GC
content strongly vary between different organisms. In animal cell
lines, a preferable GC content of 40–60% was reported [23]. In
332
Patrick Schindele et al.
a wide variety of potential target sites, and gRNAs. However,
indiscriminately selecting gRNAs can minimize and even prevent
experimental success. Thus, optimization of gRNA design is
required for improving target specificity and maximizing editing
efficiency. Pre-experimental procedures therefore often involve the
screening of different guides to determine the optimal target site.
While animal cell lines allow high-throughput screenings, evaluating gRNA-editing efficiency in other organisms can be an elaborate
process demanding longer periods for the generation of transgenic
individuals. Especially for many crops like maize and wheat, generation of transgenic plants is very time and cost intensive, and being
able to estimate gRNA efficiency before engaging in the laborious
process of transgenic plant production would be most desirable.
For this very reason, guidelines helping to define efficient gRNAs
are of tremendous importance to ensure an optimal experimental
progress. Both target site and gRNA features determine the
on-target and off-target activity of the CRISPR nuclease. Length
and sequence composition or structural features of the guide and
target, respectively, have been reported as main contributors to
overall efficiency.
1.2 Guide Length
Variation for Reduced
Off-Target Activity
The various Cas9 orthologs put different demands on PAM composition and guide length, yet, for each individual ortholog, an
optimal PAM and guide length required for maximum on-target
activity were identified [7]. Based on this observation, the most
commonly used Streptococcus pyogenes Cas9 (SpCas9) is employed
combining a 20-nt guide and the 5
0 -NGG-3
0 PAM. However, due
to the abundance of the PAM and a certain tolerance toward
mismatches, off-target activity was frequently detected in human
and animal cell lines [8, 9]. More recent studies on human 293T
cells and Drosophila reported strongly reduced off-target activity by
employing truncated guides of 17 to 18 nt in length while maintaining the editing efficiency of full-length guides [10–12]. The
strong decrease in off-target activity can presumably be explained
by a much stronger disruptive impact of mismatches on truncated
guides. Unfortunately, contradictory results have been reported in
stem cells and plants where truncated guides were less efficient
than full-length guides [13, 14]. Apart from that, full-length guides
are highly precise in plants and off-target effects only detectable
for highly similar targets or targets with PAM distal mismatches
[15–19].
1.3 Effects
of Nucleotide
Composition
and Identity on Guide
Efficiency
Concerning the effect of the nucleotide composition of the guide
and target, rather inconsistent results have been published. It is
agreed that guides having a very low or very high GC content are
less effective [20–22]. However, analysis about the optimal GC
content strongly vary between different organisms. In animal cell
lines, a preferable GC content of 40–60% was reported [23]. In
332
Patrick Schindele et al.