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Abstract <p><b>Introduction</b>. Bacteria need CRISPR-Cas systems to form an adaptive immune response necessary for protection against infectious agents. In addition, in recent years, CRISPR-Cas systems have been actively studied in the context of the development of new methods for editing the genome, which have great potential in the development of new strategies in the fight against infectious and other diseases. The mechanism involved in the formation of bacterial adaptive immunity using CRISPR-Cas systems is in the ability of bacteria to integrate fragments of foreign DNA (for example, bacteriophage DNA), which are called spacers, into their genome, which further allows bacteria to recognize and protect themselves from these infectious agents. However, not much is known about the molecular mechanisms of this process, which limits a lot the ability of scientists to understand and apply CRISPR-Cas systems. In particular, the question of the role of non-CRISPR-Cas components of bacterial cells in the formation of CRISPR adaptation remains open. Such components, for example, are intracellular nucleases, which play a role in the CRISPR adaptation process we aimed to study within our scientific project. It has been previously proven that nucleases are involved in the preparation of a foreign DNA fragment for insertion into the bacterial DNA (CRISPR cassette), which is necessary to ensure CRISPR adaptation, it has to have the certain structure and the correct length. It has been shown that aberrations in the formation of the 5'-ends of prespacers do not affect their incorporation into the CRISPR cassette and the efficiency of adaptation. <b>Material and methods.</b> Two mutant strains, Bl21-AI [delta]xni (Flap endonuclease ExoIX) and Bl21-AI [delta]polA exo (PolI), were constructed. The resulting strains were then transformed with the pCDF Cas1/Cas2 plasmid. Oligoelectroporation experiments were performed with naive adaptation induced by the addition of arabinose and IPTG. High-performance sequencing was performed using the MiniSeq™ Sequencing System (Illumina, San Diego, CA, United States). <b>Results.</b> Here we determined that DNA polymerase I likely removes extra nucleotide at the 5'-ends of spacer precursors, thereby allowing spacer precursors with extended 5'-ends to be inserted into a CRISPR cassette as spacers. <b>Conclusions.</b> The knowledge gained significantly expands the understanding of the process of CRISPR adaptation. It is of fundamental and practical significance and opens up new opportunities in the understanding and application of CRISPR-Cas systems, for instance, aiding the development of new methods to combat infectious and other diseases.</p>

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The Role of Intracellular Nucleases in the Correction of Aberrant 5'-Ends of Prespacers in the E. coli CRISPR-Cas I-E System

  • 期刊论文

  • 发表日期：

    2025年12月1日

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  • I. A. Balazs,
  • S. V. Vinogradova,
  • O. S. Musharova,
  • K. V. Severinov

摘要

Abstract

Introduction. Bacteria need CRISPR-Cas systems to form an adaptive immune response necessary for protection against infectious agents. In addition, in recent years, CRISPR-Cas systems have been actively studied in the context of the development of new methods for editing the genome, which have great potential in the development of new strategies in the fight against infectious and other diseases. The mechanism involved in the formation of bacterial adaptive immunity using CRISPR-Cas systems is in the ability of bacteria to integrate fragments of foreign DNA (for example, bacteriophage DNA), which are called spacers, into their genome, which further allows bacteria to recognize and protect themselves from these infectious agents. However, not much is known about the molecular mechanisms of this process, which limits a lot the ability of scientists to understand and apply CRISPR-Cas systems. In particular, the question of the role of non-CRISPR-Cas components of bacterial cells in the formation of CRISPR adaptation remains open. Such components, for example, are intracellular nucleases, which play a role in the CRISPR adaptation process we aimed to study within our scientific project. It has been previously proven that nucleases are involved in the preparation of a foreign DNA fragment for insertion into the bacterial DNA (CRISPR cassette), which is necessary to ensure CRISPR adaptation, it has to have the certain structure and the correct length. It has been shown that aberrations in the formation of the 5'-ends of prespacers do not affect their incorporation into the CRISPR cassette and the efficiency of adaptation. Material and methods. Two mutant strains, Bl21-AI [delta]xni (Flap endonuclease ExoIX) and Bl21-AI [delta]polA exo (PolI), were constructed. The resulting strains were then transformed with the pCDF Cas1/Cas2 plasmid. Oligoelectroporation experiments were performed with naive adaptation induced by the addition of arabinose and IPTG. High-performance sequencing was performed using the MiniSeq™ Sequencing System (Illumina, San Diego, CA, United States). Results. Here we determined that DNA polymerase I likely removes extra nucleotide at the 5'-ends of spacer precursors, thereby allowing spacer precursors with extended 5'-ends to be inserted into a CRISPR cassette as spacers. Conclusions. The knowledge gained significantly expands the understanding of the process of CRISPR adaptation. It is of fundamental and practical significance and opens up new opportunities in the understanding and application of CRISPR-Cas systems, for instance, aiding the development of new methods to combat infectious and other diseases.