Genome-wide CRISPR/Cas9 off-target profiling via Digenome-seq
- Daesik Kim
- 자연과학대학 화학부
- Issue Date
- 서울대학교 대학원
- Genome engineering; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR); CRISPR-associated protein (Cas); Whole genome sequencing (WGS)
- 학위논문 (박사)-- 서울대학교 대학원 : 화학부 생화학전공, 2016. 8. 이연.
- Targeted genome editing using engineered nuclease such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas) systems have been used in cultured cells and whole organisms for functional study and therapeutic study.
Despite broad interest in CRISPR/Cas9 mediated genomengineering, off-target effects of entire genome have not been established. Therefore, development of methods to profiling genome-wide CRISPR/Cas9 off-target effects is the major challenge in this area.
In this study, I characterize CRISPR/Cas9 off-target effect in clonal cells and bulk populations of cells. First I used Isaac variant calling program to analyze genome-wide indels in clonal cells. Second, I developed nuclease-digested genomes sequencing (digenome-seq) to profiling genome-wide CRISPR/Cas9 off-target effects in bulk populations. Using this methods, I validated off-target sites which indels were induced with frequencies below 0.1% and validated off-target effects can be avoided by replacing with modified sgRNAs. Third, I developed multiplex digenome-seq which can profiling more than ten sgRNA off-target effects in a one time. Based on multiplex digenome-seq result, I made a program for the choice of target sites and the off-target sites predictor respectively.