TY - JOUR T1 - Deeper sequencing at unexpected CRISPR/Cas9 off-target sites <em>in vivo:</em> A response to Editas, Intellia, Beacon, ToolGen and others JF - bioRxiv DO - 10.1101/154450 SP - 154450 AU - Kellie A. Schaefer AU - Wen-Hsuan Wu AU - Benjamin W Darbro AU - Diana F. Colgan AU - Stephen H. Tsang AU - Alexander G. Bassuk AU - Vinit B. Mahajan Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/08/31/154450.abstract N2 - Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness. ER -