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Temperature-Dependent Affinity Changes in Substrate Binding Affect the Cleavage Activity of BthC2c1.

Dan Wu1,2, Jieting Liu1,2, Yong Liu1,3

  • 1Heilongjiang Province Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University, Mudanjiang, 157011, Heilongjiang, China.

Protein and Peptide Letters
|January 26, 2023
PubMed
Summary

The novel Bacillus thermoamylovorans C2c1 (BthC2c1) gene editing system shows optimal DNA cleavage activity at 42°C. This temperature enhances BthC2c1-sgRNA binding affinity, offering potential for improved gene editing applications.

Keywords:
BthC2c1C2c1CRISPR-CasCpf1E. coli C43cleavage activity

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Gene Editing Technologies

Background:

  • CRISPR-Cas systems provide adaptive immunity in bacteria and archaea.
  • Cas9 and Cpf1 are established gene editing tools.
  • C2c1 is a novel endonuclease with potential for gene editing due to its small size and specificity.

Purpose of the Study:

  • To express and purify Bacillus thermoamylovorans C2c1 (BthC2c1).
  • To assemble the BthC2c1-sgRNA-dsDNA complex.
  • To investigate the effect of temperature on BthC2c1 cleavage activity.

Main Methods:

  • Prokaryotic expression and purification of BthC2c1 using GST affinity and FPLC.
  • In vitro transcription and purification of sgRNAs.
  • Assembly of BthC2c1-sgRNA-dsDNA complexes via gel filtration.
  • In vitro cleavage assays at varying temperatures.
  • Microscale Thermophoresis to determine DNA binding affinity.

Main Results:

  • BthC2c1 was successfully expressed, purified, and complexed with sgRNA and dsDNA.
  • BthC2c1 demonstrated DNA cleavage activity between 37°C and 67°C.
  • Cleavage activity and BthC2c1-sgRNA-dsDNA binding affinity were higher at 42°C compared to 37°C.

Conclusions:

  • BthC2c1 functions effectively within a specific temperature range (37°C-67°C).
  • Enhanced DNA binding and cleavage at 42°C suggest temperature-dependent mechanisms.
  • Findings provide a basis for optimizing the C2c1 gene editing system.