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Related Experiment Video

Updated: Jul 25, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Published on: February 26, 2019

Utilizing directed evolution to interrogate and optimize CRISPR/Cas guide RNA scaffolds.

Korie Bush¹, Giulia I Corsi², Amy C Yan³

¹Department of Surgery, Duke University, Durham, NC 27710, USA; University Program in Genetics and Genomics, Duke University, Durham, NC 27710, USA; Moderna Genomics, Cambridge, MA 02139, USA.

Cell Chemical Biology

|June 30, 2023

Summary

This summary is machine-generated.

Researchers developed a new method, BLADE, to create improved CRISPR-Cas9 gene editing tools. This approach enhances the editing of difficult DNA sequences by evolving new single guide RNA variants.

Keywords:

CRISPR Cas9 DNA editing SELEX aptamer guide RNA molecular evolution

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Last Updated: Jul 25, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Published on: February 26, 2019

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Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Published on: August 28, 2018

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

CRISPR-based gene editing has advanced genome engineering.
Certain DNA sequences remain difficult to target due to unproductive interactions within the single guide RNA (sgRNA) and Cas9 complex.

Purpose of the Study:

To develop a novel method for identifying diverse sgRNA variants that enhance CRISPR-Cas9 editing efficiency.
To overcome limitations in targeting challenging DNA sequences for genome engineering.

Main Methods:

A functional SELEX (systematic evolution of ligands by exponential enrichment) approach, named BLADE (binding and ligand activated directed evolution), was employed.
BLADE was used to identify and evolve sgRNA variants that effectively bind Streptococcus pyogenes Cas9 and support DNA cleavage.

Main Results:

Numerous diverse sgRNA variants with significant sequence malleability were identified.
Specific sgRNA variants demonstrated enhanced partnerships with particular DNA-binding antisense domains, leading to improved editing efficiencies at various target sites.

Conclusions:

Molecular evolution using the BLADE approach can generate CRISPR-Cas9 systems capable of efficiently editing challenging DNA sequences.
This selection strategy provides a valuable tool for creating sgRNAs with a broad spectrum of activities, making genomes more amenable to engineering.