US 11,987,790 B2
Methods for high-resolution genome-wide functional dissection of transcriptional regulatory regions
Melina Claussnitzer, Boston, MA (US); Liang He, Cambridge, MA (US); Manolis Kellis, Cambridge, MA (US); and Xinchen Wang, Cambridge, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US); and Beth Israel Deaconess Medical Center, Boston, MA (US)
Appl. No. 16/757,296
Filed by MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and BETH ISRAEL DEACONESS MEDICAL CENTER, Boston, MA (US)
PCT Filed Oct. 17, 2018, PCT No. PCT/US2018/056371
§ 371(c)(1), (2) Date Apr. 17, 2020,
PCT Pub. No. WO2019/079514, PCT Pub. Date Apr. 25, 2019.
Claims priority of provisional application 62/573,506, filed on Oct. 17, 2017.
Prior Publication US 2020/0407710 A1, Dec. 31, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 15/67 (2006.01); C12N 15/86 (2006.01); C40B 40/02 (2006.01)
CPC C12N 15/1051 (2013.01) [C12N 15/67 (2013.01); C12N 15/86 (2013.01); C40B 40/02 (2013.01); C12N 2310/20 (2017.05)] 20 Claims
 
1. A method of identifying genomic enhancer regulatory elements comprising:
fragmenting genomic DNA at accessible chromatin in a population of cells thereby generating genomic DNA fragments, wherein said fragmenting comprises transposition;
amplifying the genomic DNA fragments;
enriching the amplified genomic DNA fragments by size;
integrating the enriched fragments into a vector to obtain a vector library, wherein the vector encodes a reporter gene and the enriched fragments are integrated into an untranslated region (UTR) of the reporter gene, whereby transcription of the reporter gene results in a transcript comprising the integrated fragment sequence;
transfecting or transducing a cell line with the vector library, wherein the transcript comprising the integrated fragment sequences is expressed in the cell line; and
sequencing the transcript expressed in the cell line, whereby integrated fragments comprising enhancer activity are identified.