Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq - PubMed
4 hours ago
- #rAAV donors
- #CRISPR/Cas9
- #LOCK-seq
- The study describes a method for achieving efficient multi-kilobase knock-ins (KIs) in mice and cell lines using CRISPR/Cas9 and recombinant adeno-associated virus (rAAV) donors.
- A multi-rAAV donor approach enables precise KIs of up to 6.7 kb in one step by using three rAAVs designed to insert sequentially.
- The researchers developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets) to comprehensively characterize edited genomes, providing high coverage and detecting precise KIs, donor concatenation, and random integrations.
- LOCK-seq allows for unbiased whole-genome characterization, identifying both precise and imprecise insertions as well as non-KI allele genotypes.
- The multi-rAAV donor method is effective in cell lines, including those intolerant to plasmid DNA, and LOCK-seq facilitates reliable screening of KI clones, enhancing knock-in model creation and precision.