targeted sequencing of predicted off-target nuclease binding site #10
The mouse model of Hutchinson-Gilford progeria syndrome (HGPS) carrying the LMNA mutation G609Gﾊwas generated by Carlos L用ez-Ot地 at the University of Oviedo, Spain and kindly donated by Brian Kennedy at the Buck Institute_.ﾊThe newborn (P1) LMNAG609G/G609G (progeria) miceﾊwere used for intravenous (IV) AAV9 injection as following previous report (Lampe, S. E. G., Kaspar, B. K. & Foust, K. D. Intravenous Injections in Neonatal Mice.ﾊJoVE (Journal of Visualized Experiments)ﾊe52037ﾐe52037 (2014). doi:10.3791/52037_). The AAV mixture (AAV9-Cas9 (2x1011 GC) and AAV9-Progeria-OA (2x1011 GC) for progeria mouse) wasﾊinjected via temporal vein at P1 mouse. Afterﾊ100 days later, liver, heart, muscle, aorta,ﾊkidney and spleen were harvestedﾊfrom PBS-perfusedﾊmice. All tissues were dissected at theﾊsame amount of piecesﾊand theﾊgenomic DNAs were extracted using Blood & Tissue kit (QIAGEN #69506). The on-target and top 10 predicted off-target sites were amplified using PrimeSTAR GXL DNA polymerase and equal amount of genomic DNA collected from injection sites. Then PCR amplicons were purified using Ampure beads and 2nd round PCR to attach Illumina P5 adapters and sample-specific barcodes. The purified PCR products were pooled at equal ratio for single and/or pair-end sequencing using Illumina MiSeq at the Zhang laboratory (UCSD). High quality reads (score >23) were analyzed for insertion and deletion (indel) events and Maximum Likelihood Estimate (MLE) calculation similar to previously described methods. Briefly, raw reads with an average Phred quality score of 23 were locally aligned to their respective on or off-target sites. All reads were required to match 85% of the genomic reference region, and also span the entire 20 base-pair target region along with 5 base-pair flanking regions in both directions. Then such 30 base-pair regions were analyzed for indels, with the final indel rate calculated by using maximum likelihood estimate method similar to previously described methods that correct for background errors. As next generation sequencing analysis of indels cannot detect large size deletion and insertion events, CRISPR-Cas9 targeting efficiency and activity shown above is underestimated.