1. Outline of CRISPR-SNIPER

CRISPR-SNIPER Gene editing Services

GenAhead Bio has three associated fields, here, CRISPR/Cas9, this technology is going to form basis of our gene editing services but if you need more efficiency in genome editing, probably in knock-in (KI) experiments or SNP conversion, CRISPR-SNIPER combination would be really helpful for your purposes. SNIPER is a highly selective KI detection method by which KI process can be optimized at the beginning thereby leading to higher KI rates than some preset conditions. And also we are working with Stemgent/ReproCELL, a pioneer company of iPS, this cooperation allows us reliable genome editing on iPS cells.

CRISPR-SNIPER Gene editing Services

 

CRISPR-SNIPER Gene editing Services

  • Staffed by professional team spun out from gene editing center in Takeda Pharmaceutical Company
  • Empirically optimize knock-in condition with your own cells, with your target gene, comparing many conditions
  • Offer innovative and commercially available gene editing services
  • Enable challenging cases at high success rate including SNP conversion
  • Backed by Freedom-to-operate for research use incl drug screening
  • All-in-one service from study design to delivering gene-edited cells with QC

We were originally professional genome editing team in Takeda Pharmaceutical Company, in which we have already offered a number of services internally, such as innovative, challenging cases including SNP conversion. Now anyone are able to use this services.
 Typical genome editing service by other companies might be provided with a certain conditions e.g. same as AAVS1 KI compositions, same protocols as the other similar cells and so on. Such conditions have some success in simple genome editing. However, given that even cellular condition at the day can be an important factor for successful transfection, we believe that the best results can be obtained from empirical optimization of KI conditions, which means “in your own cells”, “with your actual gene”, looking at the KI% by SNIPER among many conditions. Our service is all-in-one package, now you have been free from troublesome design works.

 

Track Record of CRISPR-SNIPER

We had opportunities to work with some major pharmas more than 50 genome editing so far, so this portfolio might be a sort of service menu, that’s against single KO (this might be easy for all), simple deletion, stop codon insertion which is more sophisticated version of KO, GFP fusion on differentiation markers to make differentiation reporter cells, and most popular request was SNP conversion to make disease model cells. As shown in this figure, it might be evident that CRISPR-SNIPER scheme worked successfully in KI experiments. And these works were mostly done with iPS cells.

Track Record of CRISPR-SNIPER

2. Challenging Cases

2-1. SNP Conversion (most popular, Level 3)

Disease model preparation converting SNP

Nowadays many disease related mutation have been reported, about 60% of the causal mutation is point mutation. Even though it does not cause some diseases, the sensitivity of medicine could be different by SNPs just like on the top right. Therefore, on patient classification, genetic variations should be considered in clinical studies. But patient has different genetic background other than focusing SNP. So many scientists uses a very simple approach, simply changing the SNP using genome editing especially on iPS cells, because iPS cells can differentiate into any cell types with same genetic background.

Disease model preparation converting SNP

 

Bulk KI% is a Key to isolate SNP mutants in iPS cells

Bulk KI% is a Key to isolate SNP mutants in iPS cellsThe difference between SNP mutants and parental cells is only in single base, no antibiotics selection markers, therefore the only way to get these mutants would be clone pickup. How many clones do you usually pick up?
We’ve done a number of isolation studies of SNP mutants with SNIPER protocol, to look at the relationship between KI% on bulk culture and success rate in isolation. Based on these data, clear correlation between KI% and isolation study are shown. Bulk KI% is a key to isolate Homo and Hereto SNP clones. Importantly if bulk KI% is good, even with less than 30 clone pickup are enough to get Homo KI clones and hetero KI clones. In case of 1% KI, which is often seen in conventional CRISPR paper, more candidate clones would be required to find a mutant in isolation step.

 

Why so Efficient?
Why so Efficient?

 
Where these improvement come from? For the most part, they come from early estimation of KI% associated with weekly optimization, by tracking of KI% in bulk culture. This is stage 1a, which allows us the following scheduled pickup, called stage 1b. Considering from the results in previous page, we are able to know how many clones we need to pick up.
 In contrast, in conventional CRISPR scheme, you have to carry out random cloning after transfection. Random cloning means you don’t know so much about what’s going on during cloning, so you need to pickup as many as possible. It’s really laborious. If isolation failed, what we need to do is to improve experimental conditions from the beginning, but it will take very long time. So one thing note here is that, early estimation at stage 1a organizes the following things, KI%, pickup scale, and the total schedule.

 

Weekly Screen for Optimal KI Conditions (Stage1a)
Weekly Screen for Optimal KI Conditions (Stage1a)

 

Just showing SNIPER Optimization or weekly optimization. If you have a time, Step-by-Step weekly optimization will give us significant improvement of KI efficiency. On the top left, in this example, Blue is conventional CRISPR protocol and then we got >10 fold increase after 3 cycles optimization. But on the top right, just only once parallel trial using 6 wells will be often enough to select the hopeful wells. In gene B, moving away from well #3, and we should go from well #1. If you did not satisfy the KI%, we will try again changing the parameters, around 1 week.
 The parameters in 6 wells can be another sgRNA (e.g. sg1, sg2…) and also can be the amount of sgRNAs, because cutting efficiency are influenced by amount of sgRNAs. Importantly, we can use another transfection protocol (pulse1, pulse2…) and most importantly the amount of donor DNA, the direct parameter for KI.

 

Why accurate KI detection is possible with Long Donor/SNIPER ?
Why accurate KI detection is possible with Long Donor/SNIPER ?

 
Another improvement comes from the use of efficient donor. Short Donor is sometimes very good but sometimes very low as demonstrated in many papers. In contrast, long donor, which has long homology arm, has more chance to interact the genomic sequence, shows more steady KI% in our experiences. Here is such example with Presenilln SNP conversion. Regarding the quantification methods in bulk culture, long donor itself hinders correct KI event. So we developed digital PCR based highly sensitive detection system called SNIPER on which SNIPER probe hybridize actually only to correct KI sequence. Below right is an example, of SNIPER analysis with genomic DNA extracted just after KI treatment with bulk culture. By combination with reference gene probe e.g. GAPDH, actin etc., we can understand that reference positive signals are genomic DNA derived signals. After gated with reference positive, green SNIPER positive indicates correct KI.
 SNIPER allows us to use more efficient long donor thereby leading to increase in KI% and if we are able to know the KI% accurately, we can further optimize the conditions.

2-2. Knock-in (popular, Level 2)

Reporter iPSC for Differentiation Study
Reporter iPSC for Differentiation Study

 
GFP can be integrated into the genome. If a differentiation marker is targeted as the GFP acceptor, such cells will behave differentiation reporter cells. In below right, here is a review of cardiomyocyte differentiation where red fluorescence protein are fused in a differentiation marker, and GFP are fused on a maturation marker. Obviously after differentiation, red fluorescence appears first and then after maturation GFP signals appeared. This is one of the most popular application of this KI experiment. These cell lines are really useful for differentiation study for example, it’s useful to optimize differentiation protocol, to screen differentiation compound using compound library, to detect differentiated cells in organoids and so on.

 

Homo/Hetero Reporter KI in iPSCs
Homo/Hetero Reporter KI in iPSCs

 
Here is an actual KI experiment where we’ve done a GFP fusion on differentiation marker H, and after iPS clone pickup, GFP KI were checked by in-out PCR screening, on which GFP primer and gene-specific primer were used to detect the KI. KI signals are indicated by yellow arrows. And as shown in this electrophoresis profiles, we got very high KI rate, 28 KI clone in 32 clone pickup. Furthermore half of KI clone were Homo KI. We have the other examples showing very high KI rate 60 KI/60 clones and 4 KI/4 clones as high success rate.
 

2-3. Knock-out (Level 1)

Potent and Precise KO in Bulk Culture
Potent and  Precise KO in Bulk Culture

 

This is a KO experiment where we’ve done with HLA-A specific gRNA and HLA-A/B common gRNA looking at the residual copy number of target gene in bulk culture comparing a reference gene. Homology between A and B is more than 95% around here but we got very nice KO of HLA-A where HLA-B is still same (precise editing). On the other hand parallel KO compared to reference gene is possible using common gRNA.

 

Multiplex KO
Multiplex KO

 
In case of more multiple KO in bulk culture, here is an example of 5 gene destruction at once. We did not isolate the clones from this bulk culture, but this data potentially shows the possibility to save the time and effort as well as the passage number compared to Step-by-Step genome editing.

 

Advantage of CRISPR-SNIPER
Advantage of CRISPR-SNIPER

 
In summary, GenAhead Bio offers efficient genome editing strategies, sometimes by using efficient donors without interference in detection, and by optimization in accordance with actual experiments, which enables us agile approach towards success in many cases such as SNP conversion and so on.

3. License agreement with ERS Genomics

License agreement  with ERS Genomics