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┌┬5′ limitation ┌─┬PAM
5' NNNNNNNNNNNNNNNNNNNNNGG
3′ limitation┴┘
target site length 
Define the length of the target
size, sequence upstream of the PAM. :
target site 5'
limitation
(In vitro) transcription depends on a leading 'G'
(U6 promoter) or 'GG' (T7 promoter). However, it was shown
that the leading guanin(s) can also be added or substituted
at the 5' end of any sgRNA target site, hence 'NN' is set
as default.
Hwang et al., PLoS
One, (2013)
Ansai & Kinoshita, Biol. Open (2014)
:
target site 3'
limitation
A recent paper in C. elegans demonstrated that a
‚GG’ motif adjacent to the PAM could be beneficial for DSB
introduction.
Farboud & Meyer,
Genetics (2015).
:
In vitro
transcription
Select your in vitro transcription method. We will
provide the forward and reverse oligos to clone each
candidate sgRNA into the appropriate vector. If you select
"Custom" you can specify your overhangs, which will be
appended to the 5' of the sgRNA sequence.
T7 U6 Custom
fwd overhang:
rev overhang:
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┌─────max
4 MM─────┐┌─┬PAM
5' NNNNNNNNNNNNNNNNNNNNNGG
└──────────┤
max
2 MM core
max. total mismatches
Maximum number of mismatches taken into account for
prediction of potential sgRNA off-target sites. Note: more
than four mismatches in total will most likely prevent DSB
induction.
Hsu et al., Nat.
Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:
 adjust mismatch
values!
core
length
Check to enable the 'core parameter'.
More than two mismatches in the first 8-14 nucleotides
adjacent to the PAM were demonstrated to abolish Cas9
mediated DSB introduction. The 'core' is a simplified
parameter to account for these findings.
Cong et al., Science (2013)
Hsu et al., Nat.
Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:
max. core mismatches
Maximum number of mismatches to be allowed in the core
region of potential sgRNA off-target sites. Note: more than
two mismatches in total will most likely prevent DSB
induction.
Hsu et al., Nat.
Biotechnol. (2013)
Cho et al., Genome Res. (2014)
:
adjust mismatch values!
species
Choose a reference genome for sgRNA
target site evaluation.
:
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