Take the first step in the preparation of antiviral human cell lines by genomic recoding


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“Life is a complex string of codes”.

Humans have more than 20,000 genes that store all the information about life from growth to apoptosis. From discovering the structure of DNA to interpreting and writing DNA, scientists have spared no effort to explore the secrets of DNA and give scientific significance to the laws of life.

On August 2, a study published in Nature Communications provided a technical framework for the use of multi-composite base editing technology to convert the base sequence TAG containing genetic information into TAA in the human genome, and successfully achieved simultaneous editing of up to 33 gene loci in a single transfection, which in the future combined protein engineering can give cells the ability to resist viruses.

This study provides a direction and path for multiple composite editing of mammalian genomes and genome recoding to prepare human cell lines resistant to a variety of natural viruses.

Dr. Yuting Chen, Institute of Synthesis, Shenzhen Advanced Institute (former postdoctoral fellow at Harvard Medical School), Dr. Eriona Hysolli, Harvard Medical School, Dr. Anlu Chen, Harvard University, and Stephen Casper, Harvard University, are co-first authors; Professor George Church of Harvard Medical School, Chen Li Liu, Researcher of Shenzhen Advanced Institute of Synthesis, and Dr. Eriona Hysolli of Harvard Medical School are co-corresponding authors.

Writing the “Morse Code” of Living Organisms

During the war, people passed on information through the Morse code. And in the living body, there is also a string of “genetic code”.

In the double helix structure of DNA, A, T, C, and G are the bases on its structure, and through the pairing of different bases, it can eventually be arranged into 64 codons, which is known as the “genetic code” of life, including 61 codons that can encode 20 natural amino acids, and 3 codons that serve as termination signals. The achievement is to use genome recoding technology to encode the “genetic code”, which aims to give life or cells antiviral ability.

In 2016, George Church et al. proposed GP-write, which aims to shift from passive reading of genomes to active writing of genomes, using bioengineering techniques to solve many global problems facing humanity, such as viral infections, the increase in endangered species, and climate warming. In 2018, GP-write initiators proposed genome recoding to build a virus-resistant human cell line project.

Based on this, the research team proposed a potential protocol to prepare an antiviral human cell line, that is, to convert the stop codon TAG to TAA on a genome-wide scale and replace the endogenous eukaryotic release factor with an engineered mutant with selective read-through, so that the human cell line has antiviral capabilities.

At the beginning of the study, in order to quickly and accurately locate the specific location of dna codons, the research team independently developed the GRID software.

“GRIT software is like a ‘search engine’ that can search and locate the required codons on a genome-wide scale, as well as provide the guide RNA (gRNA) needed to modify codons. We used GRIT software to identify all tag codons in the human genome and synthesized gRNA that converts TAG to TAA for base editing,” Chen said.

Subsequently, they used multiple gRNA synchronous delivery and cytosine base editor (CBE) stable expression for non-targeted chain C-to-T modification, successfully realized tag conversion to TAA, and evaluated the conversion results of monoclonal cells by whole genome sequencing, RNA sequencing, and karyotype analysis, and the results showed that a transfection successfully achieved synchronous editing of 33 gene loci, and no abnormal cell gene expression and obvious chromosomal abnormalities were observed.

Genetic coding takes the first step in “antiviral”

Human genome recoding is a systematic and complex genome engineering. In this study, from identifying genome locations to multi-site gene editing, and then forming a final systematic, operational framework for each achievable piece of technology is one of the difficulties.

After four years of simulation, practice and verification, the research team successfully constructed a working framework for converting TAG termination codons into TAAs in the whole human genome, and also technically achieved editing of up to 33 gene loci in a single clone by transfection.

The study takes the first step in the preparation of a variety of natural virus-resistant human cell lines by genome recoding, preliminarily demonstrates the feasibility of TAG conversion to TAA in the human genome, and creates a record of simultaneous base editing of dozens of non-repetitive sites in the human genome, providing a working framework for large-scale engineered modification of mammalian genomes.

Today, the technology of reading DNA codes is increasingly sophisticated, but active, efficient, multi-bit writing or editing DNA codes to prepare antiviral cell lines remains a huge challenge.

“Although we have made a stage breakthrough in multi-bit gene editing technology, there is still much work to be done in the preparation of antiviral cell lines. For example, it is necessary to carry out gene editing for more loci, and optimize various technical links, protein engineering transformation, etc.,” Chen Yuting said, the research team will use genome recoding technology to further study in improving the antiviral ability of cell lines, “through this research, I hope to attract more people to pay attention to the field of large-scale genome editing or writing and recoding of antiviral cell lines, and jointly carry out the next step of research.” (Source: China Science Daily Diao Wenhui Su Qian)

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