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

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.

Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.