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Celebrating DNA Double Helix's 70th at CPL's 3rd Anniversary——Part II: DNA sequencing and gene editing


Session I: DNA Sequencing

1.Presentation by Shankar Balasubramanian

DNA: What is it Telling us?

Combining his research with DNA, Professor Balasubramanian reviewed the development in this field and looked forward to the future, especially in DNA sequencing technology. Inspired by the mechanism of DNA polymerase, Professor Balasubramanian co-invented and commercialized a brand-new high-throughput sequencing technology based on technologies such as solid-phase DNA sequencing and single-molecule DNA arrays. The new technology enormously reduced costs while increasing efficiency and it was widely used in whole genome sequencing of many species including humans. Professor Balasubramanian pointed out that genome sequencing promoted the development of genomic medicine, allowing an in-depth understanding of individual genetic information. It provided a more precise and effective method for the prevention, early screening, and treatment of diseases such as cancer.

2.Presentation by Hagan Bayley

Nanopores: DNA Sequencing and Beyond

Professor Bayley introduced the fascinating applications of nanopore sequencing technology. Unlike traditional methods which relied on chemical reactions, nanopore sequencing used electronic detection methods. When molecules such as DNA moved through the nanopores, they disrupted the current. This signal could be analyzed in real-time to determine the sequence of bases in the strands of DNA or RNA passing through the pore. It enabled long-read sequencing of single molecules without amplification and identified not only DNA, but also RNA, small molecules, or even whole polypeptide chains. By designing specific binding sites, nanopores could identify one or more target molecules. Currently, more than one million protein forms have been identified by nanopores. Small handheld instruments based on nanopores that can accurately identify, record, and calculate DNA, RNA, peptides, and small molecules were also developed.

3.Presentation by Yanyi Huang

Fluorogenic DNA Sequencing

Professor Huang shared several newly developed sequencing technologies, including a spatial transcriptomic profiling method called SPRINTseq, as well as fluorogenic error-correction code sequencing and fuzzy sequencing strategies for high-throughput sequencing instrumentation and applications. SPRINT-Seq, developed by Huang and collaborators, provides rapid sequencing speed and low cost. The technology combined the reverse-terminator sequencing-by-synthesis chemistry and in-situ rolling circle amplification, further coupled with a hybrid block coding strategy to solve the polyclonal crowdedness problem. Through fluorogenic sequencing chemistry, the sequencing accuracy was significantly increased by taking advantage of the information redundancy of multiple rounds of degenerated sequences for error detection and correction. Fuzzy sequencing, using only one round of degenerated sequencing, achieved a higher information efficiency of the reaction and a higher speed for re-sequencing applications.

Session II: Gene Editing

1.Presentation by Feng Zhang

Exploration of Biological Diversity to Improve Human Health

Professor Zhang introduced his work on human capsid assembly protein and new RNA editing systems. He described the progress in developing non-viral delivery technologies and shared the discoveries in PNMA2, which was secreted as a non-enveloped capsid and did not selectively package mRNA. After reconstituting PNMA2 capsids in vitro, the group engineered PNMA2 to bind RNA and demonstrated that the engineered PNMA2 could package and deliver mRNA into cells. In the second part, Professor Zhang introduced his work on the IscB protein family. By systematically analyzing the origin and evolutionary relationships of the CRISPR system, they found that the transposon-encoded IscB family proteins were RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely to be the ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. Using evolutionary analysis, RNA sequencing, and biochemical experiments, Zhang’s lab demonstrated that IscB used a single ncRNA for RNA-guided cleavage of dsDNA, and it could be harnessed for genome editing in human cells. This work revealed a widespread class of transposon-encoded RNA-guided nucleases, with strong developability as biotechnologies for gene editing and gene delivery.

2.Presentation by Wensheng Wei

Gene Editing and Beyond

The Wei lab focuses on developing novel gene editing tools,investigating high-throughput functional genomics and establishing novel platforms for nucleic acid therapeutics. Based on gene editing technology, they pioneered a series of novel high-throughput functional screening platforms to systematically analyze the function of protein-coding genes, non-coding RNAs, non-coding regulatory elements, and critical amino acids. These platforms include: (1) the CRISPR-based high-throughput screening technology, (2) lncRNA screening methods based on the large-fragment deletion and splice site-targeting strategies, (3) the iBAR strategy and the following BARBEKO method that enhance the efficiency and cost-effectiveness of genome-wide functional screening, (4) the PASTMUS method for scanning and identifying functional sites of proteins at amino acid resolution, and (5) the genome-wide functional amino acid mapping using ABE system. In the field of developing gene editing tools, they firstly developed a novel RNA editing technology named LEAPER and an optimized version LEAPER 2.0, which only requires the introduction of a specially designed "guide" RNA that can recruit the endogenous deaminase ADAR to achieve efficient and precise editing of specific adenosine residues on the target RNA. They further developed an efficient mitochondrial base editor called mitoBE, which has the potential to correct the majority of mitochondrial pathogenic mutations, offering an efficient and precise DNA editing tool for the treatment of mitochondrial genetic diseases. Moreover, they established an efficient in vitro technology platform for the production of high-purity circular RNA, and developed the first circular RNA vaccine against SARS-CoV-2 and its variants.