Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain

Area of Science:

• Genetics and Genomics
• Neuroscience
• Molecular Biology

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is a promising therapeutic strategy for neurodegenerative diseases such as Huntington's disease and spinocerebellar ataxias (SCAs).
• Accurate assessment of in vivo editing outcomes is crucial for the clinical advancement of CRISPR-based therapies.

Purpose of the Study:

• To evaluate the outcomes of single- and dual-guide RNA (gRNA) adeno-associated virus (AAV)-mediated CRISPR editing of the human ATXN2 gene in mouse models of spinocerebellar ataxia type 2 (SCA2).
• To establish an unbiased method for comprehensive analysis of CRISPR editing events in the brain using long-read nanopore sequencing.

Main Methods:

• Utilized polymerase-free, targeted long-read nanopore sequencing to analyze CRISPR/Cas9 editing in transgenic mouse models of SCA2.
• Assessed editing efficiency and characterized unintended genetic alterations, including AAV integration and large deletions, at the ATXN2 locus.
• Compared results with traditional PCR-based nanopore sequencing to highlight methodological biases.

Main Results:

• Achieved 10%-25% editing efficiency in the target ATXN2 gene.
• Detected unintended AAV genome integration (1%-2%) and large deletions (>150 kb) at target loci.
• Identified rearrangements of the transgenic allele (1%) and significant biases in PCR-based nanopore sequencing for AAV fragments.

Conclusions:

• Polymerase-free, long-read nanopore sequencing provides an unbiased and comprehensive approach to define CRISPR editing outcomes in vivo.
• CRISPR/Cas9 gene editing in the mouse brain via AAV delivery results in a spectrum of intended and unintended genetic modifications.
• This methodology is essential for the safety and efficacy assessment of CRISPR-based gene therapies for neurological disorders.