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Abstract <p><b>Introduction.</b> Bacteria are capable of integrating a portion of the genetic material of an invading infectious agent as a spacer into the CRISPR array. This process is called CRISPR adaptation and is necessary for bacteria to adequately respond to the same infectious agent in the future. The molecular mechanisms of CRISPR adaptation remain unclear, particularly the role of extra-CRISPR-Cas components in bacteria during prespacer generation and spacer acquisition. In this study, we developed an approach to identifying nucleases responsible for the formation of prespacer 3'-ends in vivo. <b>Materials and methods:</b> We generated a genetic construct based on the pBR322 plasmid vector with gRNAs targeting the genes encoding cellular exonucleases with 3'-5' activity in <i>E. coli</i>. This genetic construct and the pdCas9 plasmid were transformed into KD403 cells. The level of primed CRISPR adaptation was assessed using PCR and sequencing. The structure of the 3'-ends of the prespacers was assessed using the FragSeq short-fragment sequencing method using the Accel-NGS® 1 S Plus DNA Library Kit, Swift biosciences (xGenTM ssDNA &amp; Low-Input DNA Library Preparation Kit, IDT). <b>Results:</b> In the KD403 Δ<i>recB</i> strain with suppressed nucleases, a reduction in primed adaptation was detected, but sequencing revealed no disruption of the 3'-ends of the spacer precursors. <b>Conclusions:</b> Our results contribute to the understanding of the molecular mechanisms of CRISPR adaptation, which may aid in the development of CRISPR-based approaches in medicine and agriculture in the future.</p>

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Studying the Mechanisms of Prespacer 3'-End Formation during CRISPR Adaptation via Simultaneous Gene Repression Using dCas9

  • 期刊论文

  • 发表日期：

    2025年12月1日

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

摘要

Abstract

Introduction. Bacteria are capable of integrating a portion of the genetic material of an invading infectious agent as a spacer into the CRISPR array. This process is called CRISPR adaptation and is necessary for bacteria to adequately respond to the same infectious agent in the future. The molecular mechanisms of CRISPR adaptation remain unclear, particularly the role of extra-CRISPR-Cas components in bacteria during prespacer generation and spacer acquisition. In this study, we developed an approach to identifying nucleases responsible for the formation of prespacer 3'-ends in vivo. Materials and methods: We generated a genetic construct based on the pBR322 plasmid vector with gRNAs targeting the genes encoding cellular exonucleases with 3'-5' activity in E. coli. This genetic construct and the pdCas9 plasmid were transformed into KD403 cells. The level of primed CRISPR adaptation was assessed using PCR and sequencing. The structure of the 3'-ends of the prespacers was assessed using the FragSeq short-fragment sequencing method using the Accel-NGS® 1 S Plus DNA Library Kit, Swift biosciences (xGenTM ssDNA & Low-Input DNA Library Preparation Kit, IDT). Results: In the KD403 ΔrecB strain with suppressed nucleases, a reduction in primed adaptation was detected, but sequencing revealed no disruption of the 3'-ends of the spacer precursors. Conclusions: Our results contribute to the understanding of the molecular mechanisms of CRISPR adaptation, which may aid in the development of CRISPR-based approaches in medicine and agriculture in the future.