Targeted CRISPR Off-Target Analysis

The top 12 predicted off-target sites were searched using The CRISPR Design Tool^{43 (link)}. The on-target and potential off-target regions were amplified using PrimeSTAR GXL DNA polymerase from the liver DNA via IV injection and used for library construction. Equal amounts of the genomic DNA was used to amplify genomic regions flanking the on-target and top 12 predicted off-target nuclease binding sites for library construction. Next, PCR amplicons from previous step were purified using Agencourt AMPure XP, then subject to second round PCR to attach Illumina P5 adapters and sample-specific barcodes. The purified PCR products were pooled at equal ratio for single-end sequencing using Illumina MiSeq at the Zhang laboratory (UCSD). The raw reads were mapped to mouse reference genome mm9 or custom built Ai14 mouse genome using BWA^{44 (link)}. High quality reads (score >30) were analysed for indel events and Maximum Likelihood Estimate (MLE) calculation as previously described^{43 (link)}. 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.