GSE158771
HITI junction characterization
Mouse retinas and livers were injected with AAV2/8 vectors encoding for SpCas9 under control of tissue-specific promoters, as well as a donor DNA and gRNA used to perform targeted integration in the two target loci (rhodopsin for the retina and albumin for the liver) via non-homologous end joining. Retinas and livers were harvested one month after injection and DNA was extracted. PCR products comprising the 5' and 3' junctions between the endogenous loci and the inserted donor DNA were amplified and used for library generation. NGS analysis allowed characterization of the kinds and relative frequency of insertions and deletions generated at the junction points in order to better understand the precision of integration achieved by this gene editing approach. Overall design: 5' and 3' junctions were amplified as PCR products from the DNA extracted from one retina (rhodopsin) and one liver (albumin). PCR products were not amplified in negative control samples of retina and liver injected with SpCas9 and donor DNA but without a non-targeting scramble gRNA. Sequences obtained by NGS were aligned to a reference sequence of the expected genome editing outcome, which was constructed using the mouse genome and the sequence of the donor DNA. An ad-hoc algorithm was developed and used to identify insertions and deletions and calculate their relative frequency and position.