	US 12,029,760 B2
	Compositions and methods for gene editing in T cells using CRISPR/Cpf1
Yangbing Zhao, Lumberton, NJ (US); and Jiangtao Ren, Philadelphia, PA (US)
Assigned to The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Appl. No. 16/609,628
Filed by THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, Philadelphia, PA (US)
PCT Filed May 2, 2018, PCT No. PCT/US2018/030644 § 371(c)(1), (2) Date Oct. 30, 2019, PCT Pub. No. WO2018/204493, PCT Pub. Date Nov. 8, 2018.
Claims priority of provisional application 62/501,371, filed on May 4, 2017.
Prior Publication US 2020/0054679 A1, Feb. 20, 2020
Int. Cl. A61K 35/17 (2015.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/117 (2010.01); C12N 15/90 (2006.01); A61K 39/00 (2006.01); A61P 35/00 (2006.01); A61P 37/02 (2006.01)

CPC A61K 35/17 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/117 (2013.01); C12N 15/90 (2013.01); A61K 2039/5158 (2013.01); A61P 35/00 (2018.01); A61P 37/02 (2018.01); C12N 2310/122 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01)]

12 Claims

1. A method of gene editing comprising

administering to a cell:

(a) an exogenous nucleic acid comprising a stem-loop Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (st-crRNA) comprising:

(i) a crRNA comprising a target binding sequence that hybridizes to a target nucleic acid sequence in the cell,

(ii) a handle stem-loop structure attached to the 5′-end or the 3′-end of the target binding sequence, and

(iii) an additional stem-loop structure attached to the 5′ or 3′ end of the handle stem-loop structure,

wherein the st-crRNA molecule does not comprise stem-loop structures on both the 5′ and 3′ end of the crRNA, and

wherein the handle stem-loop structure is not modified with 2′-O-methyl 3′phosphorothioation (MS); and

(b) an exogenous nucleic acid encoding a Cpf1 enzyme,

wherein the st-crRNA and the Cpf1 enzyme form a RNA-guided DNA endonuclease (RGEN) complex to edit the target nucleic acid sequence.