lgRNA (Chemically ligated gRNA) is designed to improve the functions and selectivity of gRNA, particularly its efficacy, less off-target effect and stability, and the cost-effectiveness in its industrial production.
lgRNAs are very useful for therapeutic and biotechnological applications of CRISPR, particularly for multiplexing and genome scale screening with its convenience for constructing evenly distributed gRNA libraries, by bypassing carcinogenesis and immunogenicity, limits in packaging capacities, random integration into host genomes, and the complicacy and tissue infection tropisms of viral vectors and plasmids.
4. 4
Transfection of triazole ligated sg-1 (lgRNA) into a U2OS
cell line that stably expresses Cas9 results in indels at the
PPIB-targeted site.
He, K. et al. ChemBioChem 2016, 17, 1809 – 1812
Unmodified lgRNA is as Effective as sgRNA
7/1/2018
6. lgRNA vs. sgRNA vs. dual RNAs (crRNA + tracrRNA)
Efficacy in gene edition and convenience for manipulation:
sgRNA ~ lgRNA (no chemical modifications) > > dual RNAs
Capability for chemical modifications, multiplexing, and genome scale
screening:
lgRNA >> sgRNA
Advantages of chemical modifications of lgRNA: lessons from
RNA interference and antisense oligonucleotide therapeutics
̶ increase efficiency while decrease the off-target effects
̶ increase in vivo stability
̶ evade the innate immune system
̶ conjugated ligands for tissue, cell, nucleus targeting
67/1/2018
8. Catalogue Products
• lgRNA kits and custom synthesis
̶ A single dual module lgRNA optional for modifications (composed of a crRNA
and a tracrRNA ligated between 3’-end of crRNA and 5’-end of tracrRNA)
̶ A multiple module lgRNA optional for modifications (composed of a crRNA
and a multiple module tracrRNA ligated at 3’-end of crRNA and 5’-end of
tracrRNA)
• Ligated tracrRNA
• PNP kits
– Cas9-lgRNA kits
– Cas9-lgRNA library kits
7/1/2018 8
9. Nature
CRISPR/Cas9-sgRNA: Applications in Development
• Medicine
̶ Gene Therapy: genetic diseases and cancers
̶ Cures for chronic infectious diseases
̶ CAR T-cell therapy
̶ Genomic Scale screening
• Biological Research
• Animal modeling
• Agriculture
• Biofuels
• New materials
• And more
7/1/2018 9
10. The Challenges and Solution:
towards Therapeutic Applications of CRISPR/Cas9 (1)
Efficacy/Specificity:
• Off-target effects: high-frequency off-target mutagenesis induced by CRISPR-Cas
nucleases in human cells (Fu, Y. et al. Nature biotechnology 2013, 31, 822–826.
• Off-target effects: gnomically edited cells can seed tumors.
• Off-target mutations accumulate with cell proliferation for persistent Cas9
expression.
• Human pre-existing adaptive immunity against bacterial Cas9 and innate
immunity against vitro transcribed gRNA.
• Viral infectious diseases: HIV-1 escapes from the programmed CRISPR/Cas9
disruption because of non-deleterious mutations that are generated by the
cellular non-homologous end joining (NHEJ) machinery that repairs broken DNA.
• Viral vectors for expression and delivery: limits in multiplexing because of their
limited packaging capacities.
Solution:
• Chemically modified lgRNA , multiplexing and engineered CAS9
107/1/2018
11. The Challenges and Solution:
towards Therapeutic Applications of CRISPR/Cas9 (2)
Delivery:
• Efficacy and translatability of in vivo delivery methods
• Viral vectors (self-inactivating lentivirus (LVs), adenovirus, and AAV)
̶ persistent Cas9 expression after delivery may lead to undesired effects such as
higher off-target cleavage
̶ Carcinogenesis and immunogenicity of viral vectors and random integration of DNA
plasmids
̶ Limited DNA packaging capacity
̶ Tissue tropism and the difficulty to produce high-titer virus stocks
Solution:
• lgRNA with nonviral deliveries (Cas9-lgRNA PNPs ): cationic liposomes, polymers,
dendrimers, and peptides
̶ Capable of specific modifications with targeting ligands
̶ Capable of multiplexing
117/1/2018
12. Cas9-lgRNA PNPs as Human Therapeutics
lgRNAs incorporated with chemical modifications for:
Better efficiency and lower off-target effects
Better in vivo stability
Evading the innate immune system
lgRNA-ligand conjugates for tissue, cell, nucleus targeting
Low cost in industrial production
Cost efficient and more reliable multiplexing, and genome scale screening
lgRNA >> sgRNA > dual RNAs
127/1/2018
15. Synthesis of Modified Long RNAs (>100 nt): gRNA
• Limits of In-vitro transcriptions (IVT):
̶ Incompatible to non-natural RNAs
̶ Purification difficulties: DNA and proteins
̶ Heterogeneity at the 5′- and 3′- ends
̶ Have a 5′ pppG which can stimulate the cytosolic innate immune sensor
RIG-I
̶ Problematic premature transcription termination and polymerase slippage
• Chemical synthesis:
̶ the site-specific incorporation of multiple modifications at sugars, bases, or
phosphates
̶ gRNA-ligand conjugates for tissue targeting, such as GalNAc for siRNAs
̶ Robust and scalable production of highly pure sgRNAs
̶ Limits in chemical synthesis of long RNAs (>50 nt): low overall yields,
contaminations of non-full length RNAs, high cost
• Solution:
lgRNA: solid phase synthesis in combination with chemical ligations
for long modified RNAs
157/1/2018
16. Better Gene Disruption Efficiencies of CRISPR/Cas9 with sgRNA
than Dual RNAs Enhanced with Chemically Modified crRNAs
Meghdad Rahdar, et al. Synthetic CRISPR RNA-Cas9–guided genome
editing in human cells. PNAS, 2015, E7110–E7117
sgRNA > dual RNAs
167/1/2018
17. Improved Gene Disruption Efficiencies of CRIPSR/Cas9
with Chemically Modified sgRNAs
Chemically modified
guide RNAs enhance
CRISPR-Cas genome
editing in human
primary cells.
Ayal Hendel, et al.
Nature biotech, 2015,
33(9), 985-989
177/1/2018
18. Liang et al. J. Biotech. 208 (2015) 44–53
Comparison of lipid transfection of DNA, mRNA, or protein Cas9:
Cas9/gRNA RNP is the best
Cas9/gRNA RNP Delivers High On-target Efficiency but
Lower Off-target Disruptions
