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Software-based screening for efficient sgRNAs in Lactococcus lactis.

Hui Wang1, Lianzhong Ai1, Yongjun Xia1

  • 1Shanghai Engineering Research Center of Food Microbiology, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, China.

Journal of the Science of Food and Agriculture
|August 30, 2023
PubMed
Summary
This summary is machine-generated.

CRISPR gene editing relies on single-guide RNA (sgRNA) selection. This study found that sgRNA predictions in prokaryotes like Lactococcus lactis did not match experimental results, highlighting the need for empirical validation.

Keywords:
CRISPR/Cas9Lactococcus lactisgenomic engineeringsgRNA screening

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing relies on promoter and single-guide RNA (sgRNA) elements.
  • sgRNA selection is critical for precise gene targeting and editing efficiency in CRISPR systems.
  • Online prediction tools are available to predict and rank sgRNAs based on user-defined criteria.

Purpose of the Study:

  • To evaluate the accuracy of online sgRNA prediction tools in prokaryotes.
  • To compare predicted sgRNA efficiency with experimental outcomes in Lactococcus lactis.
  • To investigate the applicability of eukaryotic CRISPR findings to prokaryotic systems.

Main Methods:

  • Designed sgRNAs for Lactococcus lactis genes (ldh and upp) using the CRISPOR online tool.
  • Constructed knockout strains for selected genes to assess sgRNA efficiency.
  • Compared experimental editing efficiency with software predictions.

Main Results:

  • Experimental editing efficiencies of selected sgRNAs did not align with CRISPOR predictions.
  • Significant discrepancies were observed between predicted and actual sgRNA performance.
  • This suggests eukaryotic-centric CRISPR prediction models may not directly translate to prokaryotes.

Conclusions:

  • CRISPR sgRNA prediction software is a useful initial screening tool but requires experimental validation.
  • Established findings from eukaryotic CRISPR studies may not be universally applicable to prokaryotes.
  • Empirical testing is essential for optimizing sgRNA selection in prokaryotic gene editing.