In vivo high-throughput profiling of CRISPR–Cpf1 activity

Title
In vivo high-throughput profiling of CRISPR–Cpf1 activity
Other Titles
세포 내 대량 검증방법을 이용한 CRISPR-Cpf1 유전자 가위의 활성과 관련된 특성 확인
Author
Myungjae Song
Alternative Author(s)
송명재
Advisor(s)
고현철
Issue Date
2018-08
Publisher
한양대학교
Degree
Doctor
Abstract
Programmable nuclease is useful tools for genome editing at site-specific locus. For high efficiency of genome editing, it is important that programmable nuclease has high activity. Because Cpf1 is a newly reported nuclease protein of the class 2 CRISPR-Cas system (Zetsche, Gootenberg et al. 2015), it has limited information about Cpf1 activity profiles in mammalian cells. For this reason, Cpf1 preclude its wide use for genome editing. Here, we developed a new high-throughput method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells. A library of >11,000 guide RNA and target sequence pairs was delivered into human cells by lentiviral vectors. Subsequent delivery of Cpf1 in various methods into this cell library induced indels at the integrated synthetic target sequences, which allowed en masse evaluation of guide RNA and Cpf1 activity using deep sequencing. Regardless of delivery method of Cpf1, our hight-throughput method showed similar results and the indel frequencies measured at the integrated target sequences correlated with those at the corresponding endogenous sites. In this high-throughput approach, we determined target sequence-dependent activity profiles and newly protospacer adjacent motif sequences of Cpf1 in mammalian cells. And we found that sequence features of high activity AsCpf1 guide RNAs are distinct from those of SpCas9. Evaluation of activity at mismatched target sequences showed that Cpf1 target sequences can be divided into seed, trunk, and promiscuous regions depending on mismatch tolerability. Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.
URI
http://dcollection.hanyang.ac.kr/common/orgView/000000107524http://repository.hanyang.ac.kr/handle/20.500.11754/76074
Appears in Collections:
GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > BIOMEDICAL SCIENCE(의생명과학과) > Theses (Ph.D.)
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