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Updated: Sep 22, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Genome Editing with Cas9 in Lactobacilli.

Justin M Vento¹, Chase L Beisel^2,3,4

¹Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.

Methods in Molecular Biology (Clifton, N.J.)

|May 18, 2022

Summary

This study presents a rapid genome editing method for Lactobacillus bacteria using CRISPR-Cas9 technology. The approach facilitates genetic modification to enhance beneficial probiotic traits in these important microorganisms.

Keywords:

Genome editing Lactobacillus Recombineering Shuttle vector SpCas9

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Published on: December 11, 2020

Area of Science:

Microbiology
Bacterial Genetics
Synthetic Biology

Background:

Lactobacillus strains possess diverse metabolic and probiotic properties, making them valuable for various applications.
Genome editing is crucial for understanding genotype-phenotype relationships and improving Lactobacillus beneficial traits.
Existing CRISPR-based genome editing methods in lactobacilli show variable efficiency across different strains.

Purpose of the Study:

To develop a fast, simple, and comprehensive genome editing method for Lactobacillus species.
To provide a detailed protocol for successful CRISPR-Cas9 mediated genome editing in lactobacilli.
To offer troubleshooting guidance for common challenges in bacterial genome editing.

Main Methods:

Utilized two shuttle vectors containing a recombineering template and Streptococcus pyogenes Cas9 components.
Developed a step-by-step procedure for vector cloning, sequential transformation, and screening.
Included methods for eliminating shuttle vectors post-edit and troubleshooting escape mechanisms.

Main Results:

Successfully demonstrated a streamlined genome editing technique in Lactobacillus plantarum.
The method enables efficient genetic modification for enhancing desired bacterial traits.
Provided practical insights and troubleshooting steps for reliable genome editing.

Conclusions:

This CRISPR-based method offers a complete guide for performing genome editing in Lactobacillus.
The technique facilitates the genetic manipulation of lactobacilli for research and biotechnological applications.
The protocol aims to improve the efficiency and accessibility of genome editing in this bacterial genus.