Team-iGEM SJTU BioX 201

HP-Public-iGEM SJTU BioX 2019

Off-target incident

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.


CRISPR-Cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability. That said, off-target genetic modifications are frequent and can alter the function of otherwise intact genes. Multiple studies using early CRISPR-Cas9 agents found that greater than 50% of RNA-guided endonuclease-induced mutations were not occurring on-target.

The Cas9 guide RNA (gRNA) recognizes a 20 bp target DNA sequence, which it binds to and cleaves to alter the DNA sequence. However, target sequence binding can tolerate mismatches up to several base pairs, meaning there are often thousands of possible binding sites which present several experimental and safety concerns.

With the smooth development of CRISPR technology, a growing number of experiments are using gene editing. But in the course, off-target incident is always a problem that people cannot avoid.

How to make gene editing safer?

How to make the whole process more accurate and convenient?

In general, the public has a high degree of acceptance of microbial, non-edible animals and plants and medically-oriented human somatic cells, and has low acceptance of human embryonic gene editing and low acceptance of gene editing on edible organism. When gene editing technology does not involve the human body, most people hold a strong pragmatic standpoint and generally support gene editing of other organisms for human's purpose. For gene editing directly affecting people, the public are basically cautious.

That's where our project began—the detection of off-target effects.

Our proposal

We have designed three levels for the different needs of off-target detection. The first is real-time detection of luciferase characterization. When off-target occurs, fluorescence is generated. The second is the measure after off-target, that is, transcriptional activation produces fluorescence, and flow screening of unluminescent cells can greatly simplify subsequent detection steps. Or in another case, transcriptional activation initiates the expression of a suicide gene, and cells that are off-target lyse and die. On the third level, we implemented off-target information storage and arbitrarily read. The off-target information is stored in the plasmid containing CRISPR array by using Cas1/2, and an appropriate amount of the inducer can be added at any time to detect whether off-target has occurred or not.

Birth of Mulan

Mulan, a heroine in Chinese folktales, was adept with both the pen and the sword. This prompted us to consider developing a dual-function project just like the traditional Chinese philosophy of Yin & Yang. We have used the CRISPR/Cas system to detect, report and storage the off-target signal just like Mulan dressed like a man to scout and fight with enemies. And our project has also achieved a real-time information recording and reading system to show the gentle face of Mulan. In this way we have achieved a balance of Yin and Yang.

Mulan and society

Realising the fact that the public is generally worried about the safety of gene editing after our public survey and statistic, we decided to develop a detection and reporting system for gene editing off-target incidents to improve the safety of gene editing technology. Besides, we've learned about the huge controversy in society about gene editing through public interview and philosophy research, and therefore, we finally choose to edit simple prokaryotes that could promote to the application and acceptance of CRISPR system but with few controversies and risks. This real-time information recording and reading system can also be a way to reduce the cost of bio-storage. Moreover, through our project Mulan, we’ve built and applied a new model of human practices: "L-M-P-H-E".

You can find more detailed information in the following page: HERE


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