Establish your own bio-factory
using CRISPR-Cas9 system; A-Z
approach
Saif Aldeen AlRyalat
Introduction
• CRISPR-Cas9 system:
- Biological perspective  Bacterial immune system
- Genetic engineering perspective  Editing tool
• Genetic engineering deals with DNA, the universal language in all
livings.
Steps outline
• Choose your product
• Choose your host
• Design your CRISPR-Cas9 system
• Construct your vectors
First: Choose your product
• Several prerequisites for the
protein to produce via bacteria:
- Relatively small (preferably <40
KD)
- Without glycosylation
 Aglycosylated Abs
Ju and Jung, 2014
First: Choose your product
• Monoclonal antibodies (Abs) are recently evolving as the best
approach for several diseases like Multiple Sclerosis.
• The production and purification processes are expensive reaching
10,000$ per month.
• Using bacteria to produce Abs is an excellent alternative to provide a
significantly lower production cost.
First: Choose your product
• Aglycosylated Abs = protein part of glycosylated Ab + Peptide:NGlycosidase
• Differences between them:
- Lower cost
- Better binding to the receptor
- Reduced undesired inflammation
- Reduced circulating half-life (3wks compared with 4wks)
First: Choose your product
• Anti-angiogenic Abs approved
for age-related macular
degeneration:
- Ranibizumab (Lucentis):
Produced via Ecoli ,, 50$/dose
- Bevacizumab (Avastin):
Produced via mice ,, 2,000$/dose
Hutton D, 2014
First: Choose your product
• IgG composed of 2 Fab and 1 Fc.
• Fc mediates:
- antibody-dependent cell mediated
cytotoxicity (ADCC)
- antibody-dependent cell-mediated
phagocytosis (ADCP) via Fcγ Rs
- complement dependent cytotoxicity (CDC)
• Aglycosylated trastuzomab exhibited 160X
superior binding affinity to Fc gamma
receptor than glycosylated form.
Borrok M, 2012
Second: Choose your host
• Mainly 3 types of production
hosts:
- Mammalian (CHO)
- Eukaryotic (Yeast)
- Prokaryotic (Ecoli)
Second: Choose your host
• Factors to consider:
- Protein length
- Post-translation modification
- Cost
Demain A, 2009
Second: Choose your host
• 20 minutes doubling time.
• High cell density cultures .
• Simple laboratory setup.
• Rich complex media can be made from available and inexpensive
components.
• Fast transformation, as plasmid transformation can be performed in
less than 5 minutes.
Second: Choose your host
• Determine which Ecoli strain to
use.
• For a first expression screen,
use: BL21(DE3) or K-12 lineage:
- Missing extracellular proteases
(no protein degradation).
- Plasmid stability.
Gopal and Kumar 2013
Third: Design your CRISPR-Cas9 system
• 3 parts needed to be inserted:
- Cas9
- gRNA
- Protein of interest
Third: Design your CRISPR-Cas9 system
• Cas9 is a protein that form DSB, guided by gRNA.
• Different types depending on its bacterial origin.
• Cas9 of S.Pyogenes is a well studied protein, and the most commonly used.
• Cas9 of S.Pyogenes has 20bp gRNA and NGG Protospacer Adjacent Motif
(PAM) sequence.
• DSB caused by ionizing radiation needs 1H to be repaired, but DSB caused
by Cas9 protein needs 9H to be repaired.
Richardson C, 2016 – Nature biotechnology
Third: Design your CRISPR-Cas9 system
• gRNA design:
- 20bp length.
- adjacent to PAM sequence
(NGG) which determined by
Cas9.
- No or low off-target (small GC
bases).
• Use available software to design
your gRNA (Deskgen)
Third: Design your CRISPR-Cas9 system
• Protein of interest:
- The sequence can be taken from
genome bank (GenBank).
- Add (50-500bp) right and left
homology arms.
Ran F, 2013 – Nature protcols
Third: Design your CRISPR-Cas9 system
• Usually, repair of DSB occur by
NHEJ (90%) and HDR (10%).
• By inhibiting NHEJ, the chance of
integrating the sequence of
interest increase.
• Inhibiting DNA ligase IV (core
enzyme in NHEJ) will increase
the chance for HDR to reach 5066%.
Chu V, 2015 – Nature Biotechnology
Forth: Choose vectors
• 3 main vectors required:
- For Cas9 protein
- For gRNA
- For protein of interest
• Design your vector OR choose
from Addgene registry.
Best combination?
• Number of options when designing your system considerably high.
• Choosing the perfect combination is not possible.
 Small-scale screens can be performed either in 2-ml tubes or 96well plates.
Increase the production
1.
2.
3.
4.
5.
Change the vector
Change the strain
Change culture parameters
Co-express other genes
Change protein sequence
Gopal and Kumar, 2013
Feedback
• After optimizing previous major
steps, these strategies proposed
to overcome problems that
might be encountered:
Kolaj O, 2009 - Review
Software
• Gene optimization software:
1. Upload your protein sequence.
2. Select your expression system.
3. Specify your cloning vector.
Gopal and Kumar, 2013 – ThermoFisher scientific
Software
• Prediction of Protein Solubility in
E. coli with 94% accuracy.
• 32 Parameters taken into
account to detect the soluability.
Diaz A, 2009 - University of Oklahoma
Software
• Cello for genetic circuit design
automation:
- Order a bacteria to provide an
output based on a specific input.
- Design the genetic circuit via
Verilog programming software
and then convert it into DNA
sequence.
Thank You