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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Updated: Jul 5, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Greta Bigelyte1, Brigita Duchovska1, Rimante Zedaveinyte1

  • 1Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius LT-10257, Lithuania.

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|January 23, 2024
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Summary

New CRISPR-Cas12m proteins offer prokaryotic immunity via DNA binding, not cleavage. This discovery enables the development of compact base-editing tools for gene editing applications.

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Area of Science:

  • Molecular Biology
  • Microbial Genetics
  • Biochemistry

Background:

  • Class 2 CRISPR-Cas systems, including Cas9 and Cas12 nucleases, confer prokaryotic immunity by cleaving foreign DNA guided by RNA.
  • The diversity within the Cas12 protein family is extensive, with ongoing research to characterize novel subtypes and their functions.

Purpose of the Study:

  • To characterize a novel set of compact CRISPR-Cas12m (subtype V-M) effector proteins.
  • To elucidate the mechanism of action for Cas12m proteins in conferring prokaryotic immunity.
  • To explore the potential of Cas12m effectors in developing new gene-editing technologies.

Main Methods:

  • Biochemical assays were employed to investigate the functional properties of Cas12m effectors.
  • Cryo-electron microscopy (Cryo-EM) was used to determine the structural mechanism of DNA binding.
  • Cas12m was fused with adenine deaminase (TadA-8e) to test its efficacy in base editing.

Main Results:

  • CRISPR-Cas12m proteins provide protection against bacteriophages and plasmids through targeted DNA binding, distinct from DNA cleavage.
  • Cas12m effectors function as roadblocks, inhibiting DNA transcription and/or replication to trigger interference.
  • The Cryo-EM structure revealed the molecular basis of DNA binding and interference mediated by Cas12m.
  • Fusion of GoCas12m with TadA-8e resulted in efficient A-to-G base editing in bacterial and human cells.

Conclusions:

  • This study expands the understanding of the Cas12 protein family, highlighting a DNA-binding dependent interference mechanism in Cas12m effectors.
  • The findings reveal a novel mechanism of prokaryotic immunity mediated by DNA binding rather than cleavage.
  • Engineered Cas12m-based base editors represent a promising advancement for developing compact and efficient gene-editing tools.