# Protocol for marker-free genome editing in Saccharomyces cerevisiae using universal donor templates and multiplexed CRISPR-Cas9

## TL;DR

This paper introduces a method for marker-free genome editing in yeast using CRISPR-Cas9 and reusable components for efficient and iterative gene modifications.

## Contribution

A novel protocol for marker-free genome editing in yeast using MX6 markers and universal repair templates with CRISPR-Cas9.

## Key findings

- MX6 markers can be removed in a single CRISPR-Cas9 transformation step using universal repair templates.
- Reusable gRNA-Cas9 plasmids enable multiplexed editing and iterative use of the same selectable markers.
- PCR verification confirms the production of marker-free yeast strains suitable for further editing.

## Abstract

Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers.

For complete details on the use and execution of this protocol, please refer to Grissom et al.1

•Instructions for marker-free yeast engineering using MX6 PCR cassettes and CRISPR-Cas9•Steps for excising multiple MX6 markers in a single CRISPR-Cas9 transformation•Workflow for using reusable gRNA-Cas9 plasmids and universal repair templates in yeast•Method for iteratively reusing selectable markers after CRISPR-Cas9 marker removal

Instructions for marker-free yeast engineering using MX6 PCR cassettes and CRISPR-Cas9

Steps for excising multiple MX6 markers in a single CRISPR-Cas9 transformation

Workflow for using reusable gRNA-Cas9 plasmids and universal repair templates in yeast

Method for iteratively reusing selectable markers after CRISPR-Cas9 marker removal

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers.