plants, analysis of a significant amount of validated gRNAs revealed
a spacious GC content ranging from 30% to 80% [22]. Though
rather marginal, guides with a GC content of more than 50%
showed slightly higher efficiencies than guides with a GC content
under 50% [24]. The same was reported for Drosophila, where a GC
content over 50% within the 6 nt proximal to the PAM was
reported as beneficial, an effect that was also detected concerning
germline transmission rates of heritable mutations [12]. A similar
inconsistency is prevalent regarding nucleotide preferences. In animal and human cell lines, efficient gRNAs strongly prefer purines at
the very 3
0 -end of the guide [21]. Whereas guanine is favored at
positions 1 and 2 proximal to the PAM, thymine and cytosine are
restrictive for efficient editing [20, 25]. Cytosine is also disfavored
at position 18 distal to the PAM, however, strongly preferred at
positions 3 and 5 proximal to the PAM and as variable nucleotide of
the PAM (5
0 -CGG-3
0 PAM), respectively. Adenine preference was
detected in the midsection of the guide [20, 25]. In contrast, for
plants no significant nucleotide preferences could be validated
[22]. Merely an increased occurrence for guanine at the very
5
0 -end of the guides was detected, although this quite likely can
be attributed to the common use of the U6 small nuclear RNA
promoter constraining the first nucleotide to a guanine, at least if a
one-nucleotide “G” overhang of the gRNA is not desired. Interestingly, irregular targets starting with H nucleobases still show
comparable efficiencies in plants [24]. Analysis on target strand
preference also provides contradictory results [20, 21].
1.4 Influence
of Mismatch Nature
and Position
on Cleavage Activity
Off-target activity is largely determined by mismatch tolerance.
Early studies showed that cleavage activity of Cas9 is preferentially
abolished through mismatches in the PAM-proximal region
[2, 26]. However, studies in human cells also revealed a strong
impact of nucleotide identity on cleavage activity [9, 27]. Whereas a
G:T mismatch between gRNA and target DNA in the
PAM-proximal region only minimally affects cleavage, activity is
barely detectable for a C:C mismatch between gRNA and target
DNA [9]. The data from this study also indicate toward a significant relevance of nucleotides 5–7 proximal to the PAM with high
levels of cleavage disruption independent of nucleotide identity.
Consistent with this indication, a recent publication defined a new
core region comprising nucleotides 4–7 proximal of the PAM with
even single mismatches abolishing the majority of cleavage activity
[27]. Crucial but rather neglected features concerning on-target
and off-target activity are RNA and DNA bulges. These structures
are formed when unpaired nucleotides reside in the otherwise
consistent guide or target, respectively. In human cells, Cas9 can
tolerate DNA bulges of 1 nt all along the target sequence, though
exact positions are inconsistent between different gRNAs
[11]. RNA bulges of 1 nt can also be tolerated; however, abolish
Guidelines for gRNA Design
333
a spacious GC content ranging from 30% to 80% [22]. Though
rather marginal, guides with a GC content of more than 50%
showed slightly higher efficiencies than guides with a GC content
under 50% [24]. The same was reported for Drosophila, where a GC
content over 50% within the 6 nt proximal to the PAM was
reported as beneficial, an effect that was also detected concerning
germline transmission rates of heritable mutations [12]. A similar
inconsistency is prevalent regarding nucleotide preferences. In animal and human cell lines, efficient gRNAs strongly prefer purines at
the very 3
0 -end of the guide [21]. Whereas guanine is favored at
positions 1 and 2 proximal to the PAM, thymine and cytosine are
restrictive for efficient editing [20, 25]. Cytosine is also disfavored
at position 18 distal to the PAM, however, strongly preferred at
positions 3 and 5 proximal to the PAM and as variable nucleotide of
the PAM (5
0 -CGG-3
0 PAM), respectively. Adenine preference was
detected in the midsection of the guide [20, 25]. In contrast, for
plants no significant nucleotide preferences could be validated
[22]. Merely an increased occurrence for guanine at the very
5
0 -end of the guides was detected, although this quite likely can
be attributed to the common use of the U6 small nuclear RNA
promoter constraining the first nucleotide to a guanine, at least if a
one-nucleotide “G” overhang of the gRNA is not desired. Interestingly, irregular targets starting with H nucleobases still show
comparable efficiencies in plants [24]. Analysis on target strand
preference also provides contradictory results [20, 21].
1.4 Influence
of Mismatch Nature
and Position
on Cleavage Activity
Off-target activity is largely determined by mismatch tolerance.
Early studies showed that cleavage activity of Cas9 is preferentially
abolished through mismatches in the PAM-proximal region
[2, 26]. However, studies in human cells also revealed a strong
impact of nucleotide identity on cleavage activity [9, 27]. Whereas a
G:T mismatch between gRNA and target DNA in the
PAM-proximal region only minimally affects cleavage, activity is
barely detectable for a C:C mismatch between gRNA and target
DNA [9]. The data from this study also indicate toward a significant relevance of nucleotides 5–7 proximal to the PAM with high
levels of cleavage disruption independent of nucleotide identity.
Consistent with this indication, a recent publication defined a new
core region comprising nucleotides 4–7 proximal of the PAM with
even single mismatches abolishing the majority of cleavage activity
[27]. Crucial but rather neglected features concerning on-target
and off-target activity are RNA and DNA bulges. These structures
are formed when unpaired nucleotides reside in the otherwise
consistent guide or target, respectively. In human cells, Cas9 can
tolerate DNA bulges of 1 nt all along the target sequence, though
exact positions are inconsistent between different gRNAs
[11]. RNA bulges of 1 nt can also be tolerated; however, abolish
Guidelines for gRNA Design
333