CRISPR-Pro Versus ES Cell Targeting：Which is the Optimal Solution?

With the rapid advancement of biomedical technology in recent years, customized treatment methods such as targeted medicine, immunotherapy, and gene therapy have become the main directions of biomedical innovation - which is intimately related to the development of gene-editing technology. From the ongoing CRISPR patent battles, to the approval of the FDA on the clinical trial of human gene editing by Editas, the continued development of gene-editing technology is highly anticipated.

Modern animal model gene-editing technology can be divided into two main categories: embryonic stem cell (ESC) targeting and CRISPR-Pro editing technology. Custom genetic model types include transgenic, knockout (KO), conditional knockin (cKI), humanization, and point mutations. Given the unique advantages offered between CRISPR-Pro gene editing or ESC mediated gene targeting, which is the optimal solution? How can you choose the appropriate genetic engineering approach to generate mouse models that efficiently cater to your research needs? Let's take an in-depth comparison.

1.  Advantages and Disadvantages Analysis of the Two Technologies

ESC targeting is performed by homologous DNA recombination in mouse embryonic stem cells (ESCs). Modified ESCs are injected into the cavity of the blastocyst to form a chimeric embryo, which develops into a chimeric mouse in pseudopregnant mice. Chimeric mice are then mated with wild-type mice to pass genetic information from ESCs to offspring mice. The advantages of ESC mediated targeting include that it provides accurate gene changes without off-target effects and can perform complex genetic modifications - which is regarded as the ‘gold standard’ of the industry; notable disadvantages of the ESC mediated approach include inefficient, time-consuming process, labor-intensive, costly, and only applicable to mice. In recent years, companies have also tried various means to improve the well-established ESC gene targeting approach. Cyagen’s innovative TurboKnockout^® technology, the improved version of ES targeting, has shortened the construction turnaround time from one year to just six months – on par with CRISPR - and simultaneously reduced the overall project costs.

At present, the hottest and most widely used CRISPR-Pro system is based on Cas9 protein and guide RNA (gRNA). Cas9 contains two active sites: RuvC at the N-terminus and the HNH nuclease domain that is situated in the middle of the protein. These active sites play a coordinated role in crRNA maturation and double-stranded DNA splicing, which can cause the DNA double-strand break (DSB). After the DNA is broken, a foreign DNA fragment (homologous to the damaged DNA), also present in the nucleus, may be introduced at the target site in the genome through the homology-directed repair (HDR) mechanism to mend the damaged double-stranded DNA - thereby achieving the fragment knockin or editing. The advantages of CRISPR are its high efficiency, rapid turnaround, convenient process, low cost, and its successful application to different species; while the disadvantages include the unpredictable and uncontrollable off-target risks – making it unsuitable for complex genetic modification projects. Although a certain degree of off-target prevention can be performed through the whole-genome off-target risk calculation, for scientific research with very high rigor, off-target effects are still an inevitable problem that must be addressed.

Cyagen has analyzed over 3,400 publications by our customers, comparing the projects that use either of these two technologies (ESC Gene Targeting and CRISPR-Pro) in a variety of models: conditional knockout (cKO), knockin (KI), secondary targeting, humanization, conditional point mutation, KO-First, and other complex genetically modified projects. The results showed that the TurboKnockout^® system is used 6.4 times more often than the CRISPR-Pro approach among complex genetic modification projects, and the published articles of such complex projects generally have higher impact factors (IFs).

2. Which gene editing technology is more stable?  

There are ongoing issues surrounding the use of CRISPR, as the dispute over the rights of CRISPR core patents is still inconclusive – growing patent applications worldwide continue to build on the uncertain fate of such core patents. Given that there are thousands of CRISPR patents (including continuation patent applications, based on core patents), it is difficult for any institute or enterprise to obtain authorization for all of them via licensing. Even if all use cases are authorized by the Broad Institute, the University of California, and even Merck, it hardly avoids the risk of CRISPR patent infringement given the rapidly changing IP landscape. Moreover, the long-standing disputes in CRISPR IP may adversely affect the ability to effectively commercialize research advancements. For both organizations involved in commercial use and individuals who are concerned about IP rights, ESC targeting technology is still the most secure approach to completely avoid the risk of infringement.

3. Choosing the most suitable genetic modification method for your mouse model

As previously noted, CRISPR-Pro has no species limitation, simple operation, low cost, and short turnaround time. Therefore, it is recommended to use CRISPR-Pro technology for the construction of conventional knockout (KO) mouse models, knockin (KI) mouse models, and simple modifications in other species. However, for complex mouse model construction projects, TurboKnockout^® technology based on ESC gene targeting is still recommended.                                                                                        

ESC mediated gene targeting technology can perform a variety of complex genetic modifications accurately without any off-target risk. As the ‘gold standard’ of the industry, it can be used to build various complicated models, including: conditional knockout (cKO), knockin (KI), secondary targeting, humanization, conditional point mutation, KO-First, and more. Additionally, the improved ESC mediated TurboKnockout^® services provided by Cyagen have no associated patent issues, instead providing its own usage rights and freedom to operate. Therefore, for users who value intellectual property (IP) rights of animal models and innovations resulting from their use, it is recommended to choose TurboKnockout^® for your model generation project. As for our pharmaceutical company clients, for whom IP issues are of utmost concern, TurboKnockout^® is the preferred choice when constructing animal models used for drug development.

In summary, the two major genome modification technologies have their own advantages and disadvantages. When choosing the most suitable technology for genetically modified mice model construction, it is best to consider the comprehensive needs of your project, including: the timeline of the construction cycle, cost, off-target risk, fragment size, feasibility, species, and issues surrounding IP property rights. If you are having difficulty choosing your approach, you are welcome to call 800-921-8930 (8-6pm PST) or +1 408-969-0306 (Int'l) or email us at animal-service@cyagen.com for expert consultation at any time.

Generation of Custom Genetically Modified Animal Models:

TurboKnockout^® Knockout Mice: ES cell mediated, IP Free, As fast as 6 months, No off-target effects

CRISPR-Pro Knockout Mice: As fast as 3 months, Guaranteed germline transmitted F1 mice

Transgenic Mice: Quick turnaround time, High expression level

PiggyBac Transgenic Mice: More consistent expression, Defined region of integration, As fast as 3 months

CRISPR Knockin Mice: Large fragment up to 8kb, As fast as 4 months