I. Our Contribution - Bio Parts Addition

Our project updates the biological parts of CRISPR-Cas gene editing system for iGEM, including the sequences of LbuCas13, Cas14a1 protein, Ttcsm6 protein, sumo protein, and the expression vectors corresponding to LbuCas13, Cas14a1 protein, Ttcsm6 protein, sumo protein.

BBa_K4223008,BBa_K4223018,BBa_K4223020

In addition, we provide transcription systems for the detection of relevant target sequences used in the platform, as well as crRNA and sgRNA. (In addition, the DNA we use for Cas14a protein detection is synthetic)

crRNA: BBa_K4223012

sgRNA: BBa_K4223011

Transcription system of target RNA for Cas13a1: BBa_K4223003

Transcription system of mutated target RNA for Cas13a1: BBa_K4223010

This will facilitate future research on the drug resistance of marine microorganisms in our field by means of molecular biology, or by applying the programmability of crRNA to modify the specificity of the relevant Cas proteins to adapt and perform a thousand different nucleic acid assays.

II. Joint Manual

In order to better communicate and learn from other IGEM teams, we and the JLU-China team took the lead to write a joint handbook with other IGEM teams, detailing the whole process of different teams from the gathering of members, the initial birth of the project, communication and reflection, cooperation and working together to advance the competition. We hope that this joint manual can provide a reference for future IGEM teams to start from 0 to 1, and facilitate the project to start and finish well, and to fulfill the dream of Grand Jamboree (a lesson from the past, a lesson from the future). You can download it here. An instruction handbook for new team

This will be the framework for future exchanges and learning within and between teams.

III. Significance of the project

The qRT-PCR (quantitative reverse transcriptase-polymerase chain reaction) based assay is the current gold standard for the diagnosis of COVID-19, which has caused over 200 million infections and more than 4 million cumulative deaths in the current worldwide pandemic. Despite the high sensitivity of this method, it is still too complex and takes several hours to perform to enable rapid and immediate detection. Therefore, it is important to develop a diagnostic test strategy that is faster and easier to implement than qRT-PCR.

We exploited the ability of the Cas13a and Cas14a proteins in the CRISPR/Cas system to remain active after targeted cleavage of ssRNA (ssDNA) (Figure 1) by coupling with TtCsm6 and Csm6 activator (A4-U6 oligonucleotides) to cleave and degrade the labeled nucleic acid to generate a fluorescent signal, coupled with the base complementary pairing of This detection system has the specificity, sensitivity and efficiency that no single protein detection method has.

Based on our group, we apply it to the detection of marine pathogenic microorganisms and their drug resistance, which is beneficial to the aquaculture industry for the early detection and treatment of different pathogenic bacteria, and is also conducive to the treatment of stubborn drug-resistant bacteria when they appear, etc. At the same time, based on the programmability of crRNA (sgRNA) to modify the specificity of related Cas proteins, the technology can be applied to nucleic acid detection in various industries, different organisms and different symptoms. In other words, the system will be able to achieve targeted detection whenever nucleic acid detection is required.

Due to the tolerance of Cas13a and Cas14a proteins to 1-2 base nucleotide polymorphisms in the target sequence, the efficiency of cleavage of Cas proteins in the detection system is greatly reduced, and incorrect recognition and cleavage will also lead to "false positives" in the detection. Thus, we can assume that if the detection system contains a certain concentration of SNPS target sequences, it will be difficult to distinguish whether the detection signal comes from the target fragment that we want to track (the difference is a thousand miles). Therefore, it is essential to accurately and effectively "block" the interference of the mutated nucleic acid fragment of the target gene.

To overcome this problem and improve the specificity of the CRISPR/Cas method. We will improve the accuracy of target sequence detection and enhance the specificity of the detection system by artificially designing Peptide nucleic acids (PNA) to base complementary pair with single base mutated target sequences to achieve single base recognition detection. This solution is an improvement to the existing nucleic acid detection methods of CRISPR/Cas systems (Cas13a and Cas14a), and additionally the csm6 protein is capable of signal amplification for detection of Cas13a and Cas14a proteins (Figure 2). And use it to detect antibiotic resistance genes in Hainan Island bacteria.

The goal of achieving high precision and easy-to-use tools for CRISPR/Cas method-based nucleic acid bioassays through next-generation detection instruments provides iGEM's other teams working on CRISPR-Cas nucleic acid detection projects with the convenience of rapid and accurate detection.

IV. Hosting an online virtual meetup

HainanU-China and UESTC-BioTech are both CRISPR/Cas system based technology development teams, based on the commonality of the technologies they use, in order to seek a wider common cooperation, we jointly organized the CRISPR meetup on August 14, 2022. The CRISPR meetup was co-hosted by us and UESTC-BioTech, and teams from CPU_China, SCU-China, BIT DUT_China, HiZJU-China, NWU-CHINA-A, SCAU_China and SJTU-software were invited to share their projects, and we discussed the difficulties encountered in the process of experiments and human practices, and actively faced the difficulties in the process of human practices. We discussed the difficulties encountered in the process of experimentation and human practice, actively faced and sought solutions to them, and, during this meeting, we also built strong friendships with many teams.

A copy of the meetup brochure is attached here for all those who have visited our wiki.