Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Multiplex genome editing using CRISPR-Cas9 is powerful but hindered by issues like homologous sequence loss.
• Tandem expression of identical single-guide RNAs (sgRNAs) can lead to reduced efficiency in multiplex editing.
• Developing robust methods to screen and optimize sgRNAs is crucial for advancing genome editing technologies.

Purpose of the Study:

• To present a protocol for high-throughput screening of functional sgRNAs.
• To develop a method for generating nonrepetitive sgRNA mutants for improved multiplex genome editing.
• To enable efficient screening of sgRNA libraries for CRISPR-Cas9 applications.

Main Methods:

• Construction of a screening platform for sgRNA analysis.
• Design and construction of diverse sgRNA libraries.
• Screening of sgRNA mutants capable of interacting with the Cas9 protein.

Main Results:

• A protocol for high-throughput screening of functional sgRNAs was established.
• Nonrepetitive sgRNA mutants were generated for enhanced multiplex genome editing.
• The developed method facilitates the identification of effective sgRNAs for CRISPR-Cas9.

Conclusions:

• The presented protocol offers a solution to improve multiplex genome editing efficiency.
• This method enables the screening of functional sgRNAs, overcoming limitations of repetitive sequences.
• The protocol supports the development of more precise and efficient CRISPR-Cas9 based genome editing strategies.