Gene Editing Services
1. Insertion of large gene fragments
Normally, the rate of insertion decreases dramatically when your gene of interest (GOI) exceeds 2000 bp. As SNIPER allows optimization of gene editing conditions, it can be used in synergy with CRISPR to knock-in genes up to 7000 bp in size. This is particularly useful for tracking gene expression, as it makes the insertion of functional gene segments less challenging.
For example, you may want to insert fluorescent reporter genes to enable rapid optimization of differentiation protocols or to permit tracking of specific cell populations during differentiation. Alternatively, you may want to knock-in antibacterial resistance genes to assist cell selection.
Fig.1
2. Insertion of Biallelic Mutations
Achieving the correct disease phenotype may require the insertion of heterogenous or homogenous mutations. However, with conventional gene editing it is challenging to achieve biallelic gene modifications. Using SNIPER, gene editing conditions can be optimized to allow the creation of heterozygous or homozygous mutants for disease modelling.
Fig.2. SNIPER enables editing of both DNA strands to create a homozygous (Ho) and heterozygous (He) genotype in multiple iPSC clones.
3. Increased Screening Efficiency
Thanks to SNIPERʼs enhanced screening efficacy, the system can detect at least 30× more positive clones than conventional screening techniques.
In this way, SNIPER enhances CRISPR-Cas9 gene editing by making it easier to detect cells that contain your desired mutation. This means that CRISPR-Cas9 projects that were once unfeasible can be achieved.
Fig.3. CRISPR-SNIPER is at least 30× more efficient than CRISPR-Cas9 alone.
For SNP modification, SNIPER screening detects positive clones at a 30× higher frequency (30%) compared with conventional screening (1%).
4. Knock-out of multiple genes without increasing cell passage number
Editing multiple genes normally involves sequential gene editing experiments, each increasing cell passage number. With the CRISPR- SNIPER system you can edit up to five genes at once, thereby avoiding the effects of extended passaging, such as slow growth, formation of genetic abnormalities, and difficulties in differentiation. A further advantage of this property is the ability to assess the effect of your modification on the interaction of numerous pathway components at once.
Fig.4. Multiplex gene KO can be achieved using a range of gRNAʼs with different cleavage capabilities. By optimizing the editing efficacy for each gRNA, a KO model with multiple mutations can be created after just one round of gene editing.
5. Making challenging modifications possible
Thanks to the increased accuracy of SNIPER screening, you can now fulfil gene editing projects that may be impossible using CRISPR-Cas9 alone. This is because SNIPER combines a checkerboard of culture conditions with digital PCR to pre-screen for clones most likely to possess your desired modification. By increasing screening sensitivity, CRISPR-SNIPER makes a wider range of genome modification projects possible – including SNPs, large gene insertions and function gene insertions.
Fig.5. With SNIPER you can edit up to five genes simultaneously.
6. Achieving more accurate gene editing
It can be difficult to obtain your desired mutation if another sequence shares high homology to your GOI. By optimizing the culture conditions, guide RNA, and adding nickase to each gene editing experiment our scientists can increase the specificity of CRIPSR gene editing even further – ensuring that we only provide the cells you want.
Figure 6 illustrates that our optimized gRNA results in selective knock-down of HLA-A, whilst the HLA-B gene remains undisturbed. This is a large improvement over the results attained with non-specific RNA used in conventional gene editing, which results in knock-down of both alleles rather than one.
Fig.6. Compared with conventional gene editing techniques, SNIPER can isolate similar mutants at a high success rate.
Action Points
- Optimize gRNAs
- Set Optimal Conditions
- Perform Transfection
- Complete First Screening
Check Points
- Confirm transfection condition by bulk screening with PCR
Action Points
- Select Positive Clones
- Pick-up Positive Clones
- Perform Second screening
Check Points
- Verification of Gene Editing Success via PCR
Action Points
- Expansion of Positive Clones
- Quality Checks
- Cryopreservation of Cells
- Delivery of Cryovials
Check Points
- Verification of DNA sequence
- Screening Undifferentiated Marker Expression
- Mycoplasma Testing
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