that lead to deletion and insertion at the site. Hence, the gene or
targeted locus will be disrupted.
There are a few methods to deliver Cas9 and sgRNA effectively.
Generally, Cas9 can be delivered in the format of plasmid, mRNA,
or protein [4, 5]. Plasmid and mRNA will be transcribed and
translated into Cas9 protein in the cell cytoplasm. Subsequently,
the translated Cas9 proteins will be complexed with sgRNA. Comparatively, Cas9-sgRNA ribonucleoprotein (RNP) complexes are
more efficient in transfection and have the least off-target effects
[6]. Nonetheless, these Cas9-sgRNA RNP complexes still require
efficient delivery vehicles into the cells to achieve genomic editing.
Here, non-viral approaches are desirable as they overcome the
safety concerns posed by viral vectors [6, 7].
Scaffolds are frequently used in tissue engineering and regenerative medicine approaches to support tissue regrowth and deliver
topographical cues that can mimic the native tissue microenvironment. These scaffolds can also be modified to locally deliver cells
and therapeutics, such as proteins, small molecules, and nucleic
acids to the injured tissues. Therefore, the incorporation of
CRISPR/Cas9 systems with scaffolds offers a route to modulate
cell behavior on the scaffold. Furthermore, such scaffold-mediated
CRISPR/Cas9 systems can provide possible synergistic effects from
other factors that are co-delivered by the same scaffold.
Immobilizing proteins or nucleic acids on scaffolds have been
intensively reported [8, 9]. One method of immobilizing these
biomolecules involves surface modification of the scaffold. In this
chapter, a simple method of surface modification to functionalize
electrospun nanofibers with CRISPR/Cas9 complexes will be
described. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells.
Here, the mussel-inspired bio-adhesive coating will be used as
it is a simple and effective method to immobilize biomolecules on
surfaces. Such an approach has been demonstrated to be effective in
delivering nucleic acids in the form of siRNAs [10–12] and miRNAs [13–16], as well as Cas9-sgRNA RNP complexes [17]. As a
proof of concept, a model cell line with single copies of enhanced
green fluorescent protein (U2OS.EGFP) will be used to validate
the efficiency of genome editing.
2 Materials
2.1 Preparation
of Electrospun Fiber
Scaffolds
with Bio-adhesives
1. Polycaprolactone (PCL, M w : 80 and 40 kDa).
2. 2,2,2-Trifluoroethanol (TFE).
3. 18 mm diameter glass coverslips.
4. Carbon tape.
184
Wai Hon Chooi et al.
targeted locus will be disrupted.
There are a few methods to deliver Cas9 and sgRNA effectively.
Generally, Cas9 can be delivered in the format of plasmid, mRNA,
or protein [4, 5]. Plasmid and mRNA will be transcribed and
translated into Cas9 protein in the cell cytoplasm. Subsequently,
the translated Cas9 proteins will be complexed with sgRNA. Comparatively, Cas9-sgRNA ribonucleoprotein (RNP) complexes are
more efficient in transfection and have the least off-target effects
[6]. Nonetheless, these Cas9-sgRNA RNP complexes still require
efficient delivery vehicles into the cells to achieve genomic editing.
Here, non-viral approaches are desirable as they overcome the
safety concerns posed by viral vectors [6, 7].
Scaffolds are frequently used in tissue engineering and regenerative medicine approaches to support tissue regrowth and deliver
topographical cues that can mimic the native tissue microenvironment. These scaffolds can also be modified to locally deliver cells
and therapeutics, such as proteins, small molecules, and nucleic
acids to the injured tissues. Therefore, the incorporation of
CRISPR/Cas9 systems with scaffolds offers a route to modulate
cell behavior on the scaffold. Furthermore, such scaffold-mediated
CRISPR/Cas9 systems can provide possible synergistic effects from
other factors that are co-delivered by the same scaffold.
Immobilizing proteins or nucleic acids on scaffolds have been
intensively reported [8, 9]. One method of immobilizing these
biomolecules involves surface modification of the scaffold. In this
chapter, a simple method of surface modification to functionalize
electrospun nanofibers with CRISPR/Cas9 complexes will be
described. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells.
Here, the mussel-inspired bio-adhesive coating will be used as
it is a simple and effective method to immobilize biomolecules on
surfaces. Such an approach has been demonstrated to be effective in
delivering nucleic acids in the form of siRNAs [10–12] and miRNAs [13–16], as well as Cas9-sgRNA RNP complexes [17]. As a
proof of concept, a model cell line with single copies of enhanced
green fluorescent protein (U2OS.EGFP) will be used to validate
the efficiency of genome editing.
2 Materials
2.1 Preparation
of Electrospun Fiber
Scaffolds
with Bio-adhesives
1. Polycaprolactone (PCL, M w : 80 and 40 kDa).
2. 2,2,2-Trifluoroethanol (TFE).
3. 18 mm diameter glass coverslips.
4. Carbon tape.
184
Wai Hon Chooi et al.