Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Synthetic Biology

Background:

• Programmable gene integration is crucial for treating genetic diseases and advancing life sciences.
• CRISPR-associated transposases (CASTs) show promise for RNA-guided DNA integration but have limited efficacy in human cells.

Purpose of the Study:

• To engineer improved CRISPR-associated transposases (CASTs) with enhanced gene integration activity in human cells.
• To establish a versatile platform for directed evolution of CAST systems.

Main Methods:

• Utilized phage-assisted continuous evolution (PACE) to generate and select for improved CAST variants.
• Tested the integration efficiency and specificity of evolved CAST (evoCAST) in human cells across multiple genomic loci.

Main Results:

• Achieved over 200-fold average improvement in CAST integration activity.
• The evoCAST system demonstrated 10-30% integration efficiency for kilobase-size DNA cargoes in human cells.
• Demonstrated high specificity with undetected indels and low off-target integration at 14 diverse genomic sites.

Conclusions:

• Established a laboratory evolution platform for CAST systems.
• The evoCAST system provides a versatile and efficient tool for programmable gene integration in human cells.
• This technology has significant implications for mutation-agnostic genetic disease treatments and broader life science applications.