Chapter 13
Scaffold-Based Delivery of CRISPR/Cas9
Ribonucleoproteins for Genome Editing
Wai Hon Chooi, Jiah Shin Chin, and Sing Yian Chew
Abstract
The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian
cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are
delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral
approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for
tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local
delivery and provide possible synergistic effects with other factors such as topographical cues that are
co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to
functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface.
Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For
evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be
used to validate the efficiency of gene disruption.
Key words Gene delivery, Cas9 protein, Ribonucleoprotein, Tissue engineering, Electrospinning
1 Introduction
Clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas9 system is an efficient and simple-to-implement
genome editing technique [1, 2]. In CRISPR/Cas9-based genome
editing, two main components are needed: Cas9 protein, which is
an RNA-guided DNA endonuclease, and single-guide RNA
(sgRNA), which binds to target sequence complementarily [1–
3]. The Cas9-sgRNA complex binds to a target sequence and
induces a double-stranded break (DSB) at a specific target site.
When a template is included, the DSB may be repaired by
Homolog-Directed Repair (HDR) by inserting the template
along the break site. Without this template, the DSB can be
repaired by Non-Homologous End Joining (NHEJ) pathways
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183
Scaffold-Based Delivery of CRISPR/Cas9
Ribonucleoproteins for Genome Editing
Wai Hon Chooi, Jiah Shin Chin, and Sing Yian Chew
Abstract
The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian
cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are
delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral
approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for
tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local
delivery and provide possible synergistic effects with other factors such as topographical cues that are
co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to
functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface.
Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For
evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be
used to validate the efficiency of gene disruption.
Key words Gene delivery, Cas9 protein, Ribonucleoprotein, Tissue engineering, Electrospinning
1 Introduction
Clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas9 system is an efficient and simple-to-implement
genome editing technique [1, 2]. In CRISPR/Cas9-based genome
editing, two main components are needed: Cas9 protein, which is
an RNA-guided DNA endonuclease, and single-guide RNA
(sgRNA), which binds to target sequence complementarily [1–
3]. The Cas9-sgRNA complex binds to a target sequence and
induces a double-stranded break (DSB) at a specific target site.
When a template is included, the DSB may be repaired by
Homolog-Directed Repair (HDR) by inserting the template
along the break site. Without this template, the DSB can be
repaired by Non-Homologous End Joining (NHEJ) pathways
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183