prerequisites for designing knockout experiments. The majority of
knockout mutants are generated through nonhomologous end
joining (NHEJ)-mediated DSB repair and therefore based on the
introduction of Indel mutations. Thus, selecting pertinent target
sites within exons is of major concern. In particular, mutagenesis
within exons aims for either mutations in essential protein domains
or generation of frameshift mutations, the latter one being preferred when aiming for complete knockouts. Targeting regions too
close to the C- or N-terminus of the encoded protein is not recommended, either increasing the probability of maintaining the majority of essential domains or, if the start codon is compromised,
resulting solely in a minor displacement of transcription initiation.
Nevertheless, mutations in the encoded N-terminal region are
preferred for frameshift mutations, affecting the majority of the
coding sequences and impeding a distortion through potential
splice variants.
In the following, the design of a gRNA will be explained in
detail comprising helpful bioinformatic design tools and guide
sequence requirements based on current knowledge of gRNA
design.
2 Materials
2.1 Bioinformatic
Online Tools
1. CCTop.
2. CRISPR RGEN tools.
3. RNAfold.
2.2 Sequence
Information
1. Query sequence.
3 Methods
3.1 Guide Sequence
Selection
for CRISPR-Mediated
Mutagenesis Using
Bioinformatic Tools
The first and essential step of designing the CRISPR experiment is
the identification of the optimal guide sequence. Due to the variety
of criteria to be considered for this purpose, the use of online tools
is recommended. These tools cover the majority of the design
criteria and thereby definitely help to choose the optimal guide
sequence. To obtain an optimal consensus, the use of multiple
prediction tools is recommended. In the following, the online
tools CCTop and Cas-Designer (CRISPR RGEN tools) are
employed, both comprising a solid extent of selectable options to
prevent off-target activity and promote on-target activity.
3.1.1 CCTop
1. Go to https://crispr.cos.uni-heidelberg.de/ to open the
CCTop tool.
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Patrick Schindele et al.
knockout mutants are generated through nonhomologous end
joining (NHEJ)-mediated DSB repair and therefore based on the
introduction of Indel mutations. Thus, selecting pertinent target
sites within exons is of major concern. In particular, mutagenesis
within exons aims for either mutations in essential protein domains
or generation of frameshift mutations, the latter one being preferred when aiming for complete knockouts. Targeting regions too
close to the C- or N-terminus of the encoded protein is not recommended, either increasing the probability of maintaining the majority of essential domains or, if the start codon is compromised,
resulting solely in a minor displacement of transcription initiation.
Nevertheless, mutations in the encoded N-terminal region are
preferred for frameshift mutations, affecting the majority of the
coding sequences and impeding a distortion through potential
splice variants.
In the following, the design of a gRNA will be explained in
detail comprising helpful bioinformatic design tools and guide
sequence requirements based on current knowledge of gRNA
design.
2 Materials
2.1 Bioinformatic
Online Tools
1. CCTop.
2. CRISPR RGEN tools.
3. RNAfold.
2.2 Sequence
Information
1. Query sequence.
3 Methods
3.1 Guide Sequence
Selection
for CRISPR-Mediated
Mutagenesis Using
Bioinformatic Tools
The first and essential step of designing the CRISPR experiment is
the identification of the optimal guide sequence. Due to the variety
of criteria to be considered for this purpose, the use of online tools
is recommended. These tools cover the majority of the design
criteria and thereby definitely help to choose the optimal guide
sequence. To obtain an optimal consensus, the use of multiple
prediction tools is recommended. In the following, the online
tools CCTop and Cas-Designer (CRISPR RGEN tools) are
employed, both comprising a solid extent of selectable options to
prevent off-target activity and promote on-target activity.
3.1.1 CCTop
1. Go to https://crispr.cos.uni-heidelberg.de/ to open the
CCTop tool.
336
Patrick Schindele et al.