Team:TJU China

CRISPR system

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system, which forms the adaptive immune system in bacteria, has been modified for genome engineering. In this system, an endonuclease (Cas protein) is guided by a short sequence of RNA (gRNA) to bind to the gene of interest and cut it. Modifications of various Cas enzymes have extended CRISPR to activate/repress target genes selectively. Moreover, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA. Due to its comparative simplicity and adaptability, CRISPR system has rapidly become the most popular genome engineering approach. There are different kinds of applications of CRISPR. In our project, we are trying to improve the sensibility and gene-editing efficiency of the system itself as well as utilise it to enhance other biological approaches.

1. Heavy metal detection

Concerned about the pollutions caused by heavy metals released by manufacturers and industries to the environment including air, water and soil and causing great harm to people’s health, we are planning to utilise an engineered microorganism to detect the existence of heavy metals, aiming to develop a novel, highly sensitive and user-friendly detection device. We will engineer and construct detection and reporter circuits into E. coli and utilise CRISPR system to modify the expression of the reporter, smURFP, a kind of fluorescent protein. The change of the level of fluorescence released from smURFP will signal the existence of heavy metals. Our goal is to improve the sensitivity of the device to a great extent to guarantee the practicability and convenience of it.

2. Cell-free cancer detection

Accurate cancer diagnosis is still a challenging problem for humans now. Cell-free, circulating tumour DNA (ctDNA) can act as a noninvasive cancer biomarker, offering a potential alternative to invasive tissue biopsies. Meanwhile, researchers have taken advantage of CRISPR system to detect a defined nucleic acid. We conceive that combining the two to achieve the better cancer detection will raise far-reaching benefits for medical diagnosis, treatment and treatment response monitoring. To address problems in the diagnosis of cancer, we utilise two novel members of the CRISPR-associated protein family named Cas13a (also known as C2c2) and Cas12a (Cpf1) as detectors to indicate the existence of different cancer biomarkers by their indiscriminate single-stranded DNase and RNase activated with specific DNA and RNA, respectively[1,2]. We aim to develop a new method of diagnosis of cancer biomarkers in a high-throughput, accurate, quick and handy manner.

3. Delivery of the RNP into Hela cells

Though the CRISPR system has been widely used, the direct delivery of RNP (assembled complex of gRNA and Cas protein) into organisms is not common despite its advantages in gene-editing efficiency and specificity. The problems exist in how to enhance the delivery efficiency without affecting the gene-editing efficiency of the system. Our project focuses on the construction of our vector to efficiently transmit the Cas9 protein that silences drug-resistant genes (which is still a challenging problem in cancer treatment) into Hela cells. We choose the ABCB1 gene of P protein that is overexpressed on the surface of Hela. There are many meaningful questions waiting for us to explore, for example, how to effectively target cancer cells without being attacked by the immune system in vivo; how to ensure the stable existence of sgRNA before it works; how to ensure the stable construction of Cas9, vector and sgRNA; how to disconnect Cas9 from vector in the cell. We are going to try different kinds of two-dimensional nanomaterials, such as black phosphorus, to wrap the RNPs and deliver them into the cells through endocytosis.[3]

4. Curation of mitochondrial diseases

Mitochondrial DNA (mtDNA) mutations primarily distribute to mitochondrial diseases, and these diseases have a significant impact on human neural, audiovisual and nutritional functions. To solve this problem, we plan to utilise the CRISPR-Cas9 system to eliminate abnormal mutants and cure mitochondrial diseases. Compared with the classical methods, for example, ZFN and TALEN platforms that target by interactions of protein and DNA, the CRISPR-Cas9 system uses simple base pairing rules between engineered RNA and target DNA sites. Simple programming, unique DNA cleavage mechanisms, and the ability of multiplex target recognition enable this technique to accurately and efficiently locate, edit, modify, regulate and mark a wide range of mitochondrial genomic loci.

[1] Chen J S, Ma E, Harrington L B, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.[J]. Science, 2018:eaar6245.

[2] Li S Y, Cheng Q X, Liu J K, et al. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.[J]. Cell Research, 2018.

[3] Yue H, Zhou X, Cheng M, et al. Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing.[J]. Nanoscale, 2017, 10(3).