한빛사
>
한빛사논문
한빛사논문
- 조회425
Nat. Protoc., Jun 06 2024, | https://doi.org/10.1038/s41596-024-01003-0
Molecular recording using DNA Typewriter
Hanna Liao1,2,7, Junhong Choi 1,3,7* & Jay Shendure1,4,5,6*
1Department of Genome Sciences, University of Washington, Seattle, WA, USA.
2Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.
3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
4Howard Hughes Medical Institute, Seattle, WA, USA.
5Brotman Baty Institute for Precision Medicine, Seattle, WA, USA.
6Allen Discovery Center for Cell Lineage Tracing, Seattle, WA, USA.
7These authors contributed equally: Hanna Liao, Junhong Choi.
*Corresponding authors
Correspondence to Junhong Choi or Jay Shendure.
Abstract
Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell's DNA, marking the genome with information about a cell's history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the 'substrate' to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data ( https://doi.org/10.6084/m9.figshare.22728758 ). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4-6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.
논문정보
- Review Paper
- 2024년 06월 (BRIC 등록일 2024-06-12)
- 국외 연구진
- 생명과학 > 분자생물학
댓글 작성 로그인
팝업 닫기관련 링크
연구자 키워드
소속기관 논문보기
관련분야 논문보기
해당논문 저자보기
