The development of genome editing tools as powerful techniques with versatile applications in biotechnology and medicine: CRISPR/Cas9, ZnF and TALE nucleases, RNA interference, and Cre/loxP

Abstract

AbstractThe huge progress in whole genome sequencing (genomic revolution) methods including next generation sequencing (NGS) techniques allows one to obtain data on genome sequences of all organisms, ranging from bacteria to plants to mammals, within hours to days (era of whole genome/exome sequencing) (Goodwin et al. in Nat Rev Genet 17:333–351, 2016; Levy and Myers in Annu Rev Genomics Hum Genet 17:95–115, 2016; Giani et al. in Comput Struct Biotechnol J 18:9–19, 2020). Today, within the era of functional genomics the highest goal is to transfer this huge amount of sequencing data into information of functional and clinical relevance (genome annotation project). The World Health Organization (WHO) estimates that more than 10,000 diseases in humans are monogenic, i.e., that these diseases are caused by mutations within single genes (Jackson et al. in Essays Biochem 62:643–723, 2018). NGS technologies are continuously improving while our knowledge on genetic mutations driving the development of diseases is also still emerging (Giani et al. in Comput Struct Biotechnol J 18:9–19, 2020). It would be desirable to have tools that allow one to correct these genetic mutations, so-called genome editing tools. Apart from applications in biotechnology, medicine, and agriculture, it is still not concisely understood in basic science how genotype influences phenotype. Firstly, the Cre/loxP system and RNA-based technologies for gene knockout or knockdown are explained. Secondly, zinc-finger (ZnF) nucleases and transcription activator-like effector nucleases (TALENs) are discussed as targeted genome editing systems. Thirdly, CRISPR/Cas is presented including outline of the discovery and mechanisms of this adaptive immune system in bacteria and archaea, structure and function of CRISPR/Cas9 and its application as a tool for genomic editing. Current developments and applications of CRISPR/Cas9 are discussed. Moreover, limitations and drawbacks of the CRISPR/Cas system are presented and questions on ethical concerns connected to application of genome editing tools are discussed.