6. Upload the trace files and the guide RNA sequence (20 nt) into
the TIDE web tool (available at http://tide.nki.nl and https://
deskgen.com) and perform the analysis with default
parameters.
Use the TIDE assay to compare the efficiency of the different
guides and select the best experimental conditions for further
experiments. An example of TIDE analysis is provided on the
TIDE website.
3.3 Design
of Plasmid Donor
Several types of DNA repair template can be utilized in
HDR-mediated CRISPR/Cas9 gene editing. Donor plasmids,
with homology arms of 300–1000 bp allow for large insertions in
the 1–2 kbp range. Other types of DNA repair templates such as
linear DNAs with short homology arms obtained by PCR or gene
synthesis can also be used for inserting fluorescent labels or other
protein tags such as the Halo or Snap tags [22, 23]. Linear DNA,
however, may concatemerize before insertion [24], which may
perturb gene expression or make the modification unstable during
cell replication. Different types of donor DNA will give similar or
different KI efficiencies depending on the gene targeted and cell
line used and may be tested if needed.
This section details the design and preparation of the donor
plasmid which requires the introduction of the coding sequence for
the insertion flanked by two homology arms of a few hundred bases
(typically between 0.3 and 1.0 kbp).
1. Define the DNA fragment that you wish to introduce, which in
this example case consists in the eGFP-P2A-PURO coding
sequence where eGFP, the 2A self-cleaving peptide (P2A),
and the puromycin N-acetyl transferase coding sequence are
separated by flexible glycine/serine-rich linkers (Fig. 4). References [25, 26] provide useful information on the use of 2A
peptides in a polycistronic vector and the design of protein
linkers, respectively. Note that when using 2A self-cleaving
peptide, antibiotic resistance is expressed from the target
gene, which minimizes the selection of clones with
non-targeted integration. However, in some cases, expression
levels of the target gene may be insufficient to provide antibiotic resistance, and it may be necessary to provide antibiotic
resistance from a constitutive promoter; higher numbers of
clones with random integration of the donor plasmid into the
cell genome may be isolated [27].
2. Retrieve the sequence of a fragment encompassing upstream
and downstream homology arms of approximatively 700 bp
and replace the STOP codon of the GOI by the eGFP-P2APURO coding sequence. Carefully verify that the eGFP-P2APURO coding sequence is in frame with the last exon of the
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
49
the TIDE web tool (available at http://tide.nki.nl and https://
deskgen.com) and perform the analysis with default
parameters.
Use the TIDE assay to compare the efficiency of the different
guides and select the best experimental conditions for further
experiments. An example of TIDE analysis is provided on the
TIDE website.
3.3 Design
of Plasmid Donor
Several types of DNA repair template can be utilized in
HDR-mediated CRISPR/Cas9 gene editing. Donor plasmids,
with homology arms of 300–1000 bp allow for large insertions in
the 1–2 kbp range. Other types of DNA repair templates such as
linear DNAs with short homology arms obtained by PCR or gene
synthesis can also be used for inserting fluorescent labels or other
protein tags such as the Halo or Snap tags [22, 23]. Linear DNA,
however, may concatemerize before insertion [24], which may
perturb gene expression or make the modification unstable during
cell replication. Different types of donor DNA will give similar or
different KI efficiencies depending on the gene targeted and cell
line used and may be tested if needed.
This section details the design and preparation of the donor
plasmid which requires the introduction of the coding sequence for
the insertion flanked by two homology arms of a few hundred bases
(typically between 0.3 and 1.0 kbp).
1. Define the DNA fragment that you wish to introduce, which in
this example case consists in the eGFP-P2A-PURO coding
sequence where eGFP, the 2A self-cleaving peptide (P2A),
and the puromycin N-acetyl transferase coding sequence are
separated by flexible glycine/serine-rich linkers (Fig. 4). References [25, 26] provide useful information on the use of 2A
peptides in a polycistronic vector and the design of protein
linkers, respectively. Note that when using 2A self-cleaving
peptide, antibiotic resistance is expressed from the target
gene, which minimizes the selection of clones with
non-targeted integration. However, in some cases, expression
levels of the target gene may be insufficient to provide antibiotic resistance, and it may be necessary to provide antibiotic
resistance from a constitutive promoter; higher numbers of
clones with random integration of the donor plasmid into the
cell genome may be isolated [27].
2. Retrieve the sequence of a fragment encompassing upstream
and downstream homology arms of approximatively 700 bp
and replace the STOP codon of the GOI by the eGFP-P2APURO coding sequence. Carefully verify that the eGFP-P2APURO coding sequence is in frame with the last exon of the
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
49