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

The 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.

Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism

“…One of the first-exon deletions was lethal in utero (Leiper et al 2007 ; Breckenridge et al 2010 ), whereas all the other knock-out models, including the one used in this study, had no detrimental effect on embryonic development. Generating knock-out constructs entails the risk of mutagenic and/or off-target effects and its precision highly depends on the particular genome engineering approach (Schulze and Lammers 2021 ). First-exon deletion may have interfered with neighboring genes essential for embryonic development, leading to impaired embryo implantation and death (Breckenridge et al 2010 ).…”

RNA-Mediated Nuclease Genome Editing System Type II: A Mini Review

“…The HNH domain cleaves the 20-mer complementary target sequence to the sequence in the spacer unit of crRNA. The domains are characterized on the basis of their structure in the helix DNA (Watson's DNA structure) (Schulze et al, 2020). The active site of the HNH domain has 2 Beta-Alpha metal folds and these folds in the HNH domain show a similarity with the other nuclease such as phage T4 endonuclease 7 and Vibrio vulnificus nuclease.…”

CRIPSR Case System: Biological Role in Bacterial Virulence, Genome Editing and in Antimicrobial Resistance

“…Cas9 act as nuclease. CRSIPR Cas technology can be used for in vitro genome editing (Schulze and Lammers, 2020). Scientists synthesize a small fragment of RNA with short 'guide' sequence to that binds with a particular target sequence in genome.…”