| Abstract [eng] |
CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated protein) is a gene editing system derived from prokaryotic organisms that can recognize and degrade nucleic acid sequences in a guide-RNA dependent manner. The system is represented by a nucleoprotein effector complex that consists of Cas proteins and a guide RNA that targets the system to the DNA fragment of interest. Based on the structure of the effector complex, as well as signature proteins CRISPR-Cas systems can be divided into two major classes and a variety of types and subtypes. Class 1 systems are characterized by an effector complex that consists of multiple proteins, while class 2 systems have an effector complex with a single effector protein. Type IV CRISPR-Cas systems belong to class 1, and have a compact, crRNA-guided multi-subunit effector complex. Due to their ability to bind and cleave predetermined regions of DNA or RNA, CRISPR-Cas systems are often used in gene editing. Beyond nucleic acid cleavage, deactivated CRISPR-Cas complexes, sometimes fused to regulatory domains, can be used for regulation of gene expression in systems such as CRISPRi, CRISPRa or CRISPR-mediated epigenetic editing. Despite the development of versatile tools for genome editing based on CRISPR-Cas systems, potential applications of CRISPR editing in human cells is limited by low cargo capacity of commonly used viral vectors, such as AAV. However, there is a possibility to adapt type IV CRISPR-Cas systems for use with low cargo capacity vectors, as their effector complexes are compact, compared to the common CRISPR-Cas effectors such as Cas9. Another advantage of type IV CRISPR-Cas over conventionally used CRISPR-Cas systems is their multi-subunit effector complex. Type IV system subunits can be fused with regulatory domains and used in CRISPRi, CRISPRa or CRISPR epigenetic editing, since it allows for the development of different Cas protein–effector domain fusion strategies. This research work describes the adaptation of a type IV CRISPR-Cas system for genome editing, which includes construction of a plasmid vector for expression of a type IV CRISPR-Cas system in human cells, development of a minimal CRISPR-Cas type IV system and development of next-generation genome editors based on type IV CRISPR-Cas. The research carried out can help to establish class 1 type IV CRISPR complexes as viable tools for eukaryotic gene editing. |
