CRSIPR/Cas9: Magic scissors for genome editing

Abstract

The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing.” Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using this strategy, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true. The CRISPR sequence was first discovered in E. coli. by Japanese molecular biologist Yoshizumi Ishino in 1987, and the Spanish microbiologist Francisco Mojica further discovered unique non-repetitions in CRISPR in 2003. Later on, it has been realized that CRISPR is an adaptive immune system derived from bacteria. CRISPR can identify (via crRNA) viruses that invade bacteria, and use a special nuclease (Cas9 protein) to degrade the DNA sequence of the invading viruses, thereby protecting bacteria from viral invasion. Emmanuelle Charpentier discovered that there are some new small RNA molecules in Streptococcus pyogenes in the process of studying the regulatory RNA of Streptococcus pyogenes, whose genetic code partially matches the CRISPR sequence. Furthermore, they have discovered that these unknown RNA molecules (later known as trans-activated CRISPR RNA, tracrRNA) can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active RNA (crRNA). The first comprehensive analysis of the principles of CRISPR/Cas9 gene editing came from the collaboration of Emmanuelle Charpentier and Jennifer Doudna. They found that using recombinant S. pyogenes Cas9 protein and in vitro transcribed crRNA and tracrRNA, either purified DNA or E. coli gene could be efficiently edited. In addition, they proved that both crRNA and tracrRNA are necessary for Cas9 to be functional, and that the two RNAs can also function in vitro when they are fused into a single guide RNA (sgRNA). Shortly, the research group from MIT reported for the first time that CRISPR/Cas9 gene editing technology can be applied to gene editing in mammalian and human cells, thus promoting the explosive development of this technology. Using the CRISPR gene editing method, researchers can theoretically direct Cas9 nuclease to perform gene editing at any desired gene locus by designing different sgRNAs. Due to the simplicity, low-cost, and efficiency of this method, CRISPR has rapidly developed into the most mainstream gene editing technology, which is used to efficiently and accurately change, edit or replace plant, animal and even human genes. CRISPR/Cas9 gene editing technology has shown a great potential for biomedical applications, but it still associated with challenges that include biosafety, off-target effects of gene editing. For example, the human body may have an immune response to the gene-editing nuclease Cas9 derived from bacterial; it is very important to efficiently and safely introduce the CRISPR/Cas9 gene-editing system into living cells or in vivo . Viral vectors have been widely used in human gene therapy, but their loading capacity for Cas9 is greatly limited. Therefore, the development of new CRISPR/Cas9 delivery systems is particularly important for in vivo gene editing and its clinical transformation applications. To reduce off-target effects of CRISPR/Cas9 genome editing, site-directed mutation of Cas9 protein or the development of new gene editing technologies (such as base editing) are highly appreciated for promoting this technology into clinic.