The liposome is an ideal drug delivery carrier with characteristic lipid bilayers that resemble the cell plasma membrane and high biosecurity. They can incorporate a variety of drug candidates including anti-cancer formulations, nucleic acid and antigens. Liposomes are the most widely used carriers for delivering CRISPR/Cas9 due to their capacity to extend blood circulation time and prevent the enzymatic digestion in the serum. Besides, cationic liposomes can effectively avoid the electrostatic repulsion of negatively charged cell membranes and improve cellular uptake and endosomal escape. https://www.creative-biolabs.com/lipid-based-delivery/

1. Creative Biolabs|Lipid-based Drug Delivery
LIPOSOMES
FOR GENE
EDITING
Delivery of CRISPR/Cas9
An expert in Lipid-based Drug
Delivery Services

2. Creative Biolabs|Lipid-based Drug Delivery
Contents
1 2 3 4 5
Background CRISPR/Cas9 Liposomal
Delivery of
CRISPR/Cas9
Liposome
Surface
Modifications
Recent Studies
on Liposomal
Delivery of
CRISPR/Cas9

3. In recent years, new gene-editing techniques have been developed and applied to various
diseases treatment. Among them, clustered regularly interspaced short palindromic repeats
(CRISPR) appears to be the most suitable for preclinical and clinical applications compared to
other gene-editing technologies due to its high gene editing efficiency and simultaneous
multiple loci editing.
However, several barriers limited the direct use of CRISPR in gene therapy. First, CRISPR/Cas9
could be digested by endonuclease in the serum. Second, CRISPR/Cas9 is anionic, which
reduces cell uptake efficiency due to electrostatic repulsion. Therefore, it is necessary to select
carriers that meet the following key features for efficient CRISPR delivery: (1) Prolonged blood
circulation time; (2) Efficient delivery into target tissues; (3) High cellular uptake and efficient
endosomal escape.
Among the existing delivery carriers, liposomes with characteristic lipid bilayers that resemble
the cell plasma membrane are the most widely studied gene carriers due to their excellent
biodegradability and biocompatibility. In the liposomal CRISPR delivery system, many
parameters including size, surface charge, PEGylation, and targeting ligands can be adjusted as
need. Moving forward, liposomes will deliver their promise for further applications providing
their serum stability, higher endosomal escape, and transfection efficiency.
Creative Biolabs|Lipid-based Drug Delivery
Background

4. Creative Biolabs|Lipid-based Drug Delivery
• Fuse crRNA and tracrRNA into single-guide
RNA (sgRNA) to improve the stability
• Base pairing between the 5‘ -terminal 20 nt of
sgRNA and target DNA
• Cas9 recognizes the PAM sequence at the 3’ –
terminal of the target DNA
• Double-strand break (DSB) after CRISPR/Cas9
cleaves the target DNA
CRISPR/Cas9

5. Creative Biolabs|Lipid-based Drug Delivery
• dCas9 only retained the ability to be guided
into the genome by gRNA, and the cleavage
enzyme activity was completely lost
• The Cas9nickase is a mutant form of the Cas9 nuclease
where one of its cleavage domains has been disabled to
minimize the off-target effects of wild type Cas9
Variants of Cas9

6. Creative Biolabs|Lipid-based Drug Delivery
The liposome is an ideal drug delivery carrier with characteristic lipid
bilayers that resemble the cell plasma membrane and high biosecurity.
They can incorporate a variety of drug candidates including anti-cancer
formulations, nucleic acid and antigens. Liposomes are the most widely
used carriers for delivering CRISPR/Cas9 due to their capacity to extend
blood circulation time and prevent the enzymatic digestion in the serum.
Besides, cationic liposomes can effectively avoid the electrostatic
repulsion of negatively charged cell membranes and improve cellular
uptake and endosomal escape. Different structures of liposomes such as
lipoplexes, stable nucleic-acid-lipid particles (SNALPs), lipopolyplexes,
and membrane/core nanoparticles (MCNPs) can provide encapsulation
of CRISPR/Cas9 plasmid, Cas9 mRNA with sgRNA or Cas9–gRNA
ribonucleoprotein. The flexibility of liposomes also allows the many
parameters to be adjusted as needed, including the degree of
PEGylation, targeting ligands, and surface charge.
Liposomal Delivery of
CRISPR/Cas9

7. Creative Biolabs|Lipid-based Drug Delivery
Modify targeting ligands on
liposomes to enhance cellular
uptake further increases the
gene-editing efficiency
Adjust surface charge of
liposomes to control cell
uptake, endosomal escape,
blood circulation and tissue
accumulation
PEGylation to increase
structural stability and
circulation time of
liposomes
Liposome Surface
Modifications

8. Creative Biolabs|Lipid-based Drug Delivery
Name Lipid Form of CRISPR Ligands Disease model
Liposome-templated hydrogel
nanoparticles (LHNPs) DOTAP Cas9 protein/Minicircle DNA iRGD, mHph3 Cancer
Liposome-coated mesoporous
silica nanoparticle (lipoMSN) DOTAP Cas9/gRNA RNPs / Cardiovascular disease
Lipofectamine 2000
Cationic
liposome Cas9/gRNA RNPs / Genetic deafness
Lecithin-based liposomal
nanocarrier particle (NL) Lecithin Cas9/gRNA RNPs PEI Type 2 diabetes mellitus
Recent Studies on Liposomal
Delivery of CRISPR/Cas9

9. Creative Biolabs|Lipid-based Drug Delivery
Liposome Design
• Formulation
• Structure
• Function
• Cargo
Liposome Production
• Encapsulation
• Combination of Nanoparticles &
Materials
• Surface Charge Adjustment
• PEGylation
• Targeting Ligands Modification
• Controlled Release
• Other Surface Modifications
Liposome Analysis and
Characterization
• Basic characterization Analysis
• Formulation Stability Monitoring
• Functional Verification & Analysis
• Other custom Services
Creative Biolabs’ Services
of Liposome

10. Creative Biolabs|Lipid-based Drug Delivery
Our liposomes custom services can achieve prolonged blood circulation
time, high cellular uptake, efficient endosomal escape and functional
modification for liposomal delivery of CRISPR/Cas9. Different structures
and ligands are optional according to requirements. Our services include
liposome structure design, liposome encapsulation, liposome ligands
modification and characterization. We can also provide strategies to
combine liposomes with nanoparticles or other materials.
Features of our Liposomes Custom Services:
• High biocompatibility, low toxicity
• High cellular uptake and endosomal escape efficiency
• Encapsulation of different form of CRISPR/Cas9
• Multi-functionalization
As a liposome contract manufacturer, we are
making high-end liposomal products for customers.
Creative Biolabs can customize the design & production
of a liposomal product for you completely in line with your
requirements. Our ground- breaking research, objectified
working processes, and state-of-the-art equipment all mean
that we can develop distinctive liposomal formulations
and products. Our liposomes custom services offer genuine
phospholipid bilayered vesicles containing the active
substance. We can formulate liposomes for any active
substance. Besides, our unique liposome products are subject
to stringent quality control, and we can guarantee that our
liposomes retain their quality and efficacy during storage. For
more details about our services, please contact us directly.
Creative Biolabs’ Services
of Liposome

11. CONTACT
SUITE 203, 17 Ramsey Road,
Shirley, NY 11967, USA
Tel: 1-631-871-5806 (USA)
44-207-097-1828 (Europe)
Fax: 1-631-207-8356
Email: info@creative-biolabs.com