Safe harbor loci

Abstract

Provided herein are safe harbor loci and methods for identifying and using safe harbor loci to engineer cells to express transgenes. The safe harbor loci exhibit increased transgene knock-in efficiency and allow for increased, stable expression of transgenes in engineered cells. Guide ribonucleic acids (gRNAs) may be used for insertion of transgenes in the safe harbor loci.

1 . A method of editing a cell having chromosomal DNA, comprising inserting at least one sequence comprising a transgene within a safe harbor locus in the chromosomal DNA of the cell, wherein the safe harbor locus is at any one or more of an sgRNA target loci selected from: chr11:128340000-128350000, chr15:92830000-92840000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr16: 11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, chr9:7970000-7980000, APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28. 
     
     
         2 . The method of  claim 1 , wherein the sgRNA target locus is chr11:128340000-128350000 or chr15:92830000-92840000. 
     
     
         3 . The method of  claim 1 , wherein the sgRNA target locus comprises an integration site comprising a start coordinate of chr11:128342576. 
     
     
         4 . The method of  claim 1 , wherein the sgRNA target locus comprises an integration site comprising a start coordinate of chr15:92831131. 
     
     
         5 . The method of  claim 1 , wherein the at least one sequence is operatively linked to an endogenous promoter. 
     
     
         6 . The method of  claim 1 , wherein the at least one sequence is operatively linked to an exogenous promoter. 
     
     
         7 . The method of  claim 6 , wherein the exogenous promoter is an EF1α promoter. 
     
     
         8 . The method of  claim 1 , wherein the cell is a natural killer (NK) cell, an induced pluripotent stem cell (iPSC), a human pluripotent stem cell (HSPC), a T cell, a CD8+ T cell, or a T cell progenitor cell. 
     
     
         9 . The method of  claim 8 , wherein the cell is a T cell, a CD8+ T cell, or a T cell progenitor cell. 
     
     
         10 . The method of  claim 8 , wherein the cell is a CD8+ T cell. 
     
     
         11 . The method of  claim 13 , wherein the transgene encodes a protein, a therapeutic agent, a cancer-specific therapeutic agent, a T-cell receptor (TCR), a Prime Receptor (primeR), or a chimeric antigen receptor (CAR). 
     
     
         12 . The method of  claim 1 , wherein the transgene encodes a chimeric antigen receptor (CAR). 
     
     
         13 . The method of  claim 1 , wherein the transgene encodes a T-cell receptor (TCR). 
     
     
         14 . The method of  claim 1 , wherein the transgene encodes a Prime Receptor (primeR). 
     
     
         15 . The method of  claim 1 , wherein the transgene encodes an shRNA, an miRNA, or an siRNA. 
     
     
         16 . The method of  claim 1 , wherein the at least one sequence is inserted using a homology-directed repair or a homology independent targeted insertion. 
     
     
         17 . The method of  claim 1 , wherein the at least one sequence is inserted using one or more guide ribonucleic acids (gRNAs) and one or more Cas9 endonucleases, optionally wherein the one or more gRNAs comprises any one of SEQ ID NOS: 1-120. 
     
     
         18 . The method of  claim 17 , wherein the one or more gRNAs comprises SEQ ID NO: 94. 
     
     
         19 . The method of  claim 17 , wherein the one or more gRNAs comprises SEQ ID NO: 102. 
     
     
         20 . A guide ribonucleic acid (gRNA) for editing a cell at a safe harbor locus, wherein the gRNA comprises any one of SEQ ID NOS:1-120. 
     
     
         21 . An ex vivo method of obtaining an engineered cell or population thereof, comprising:
 a. obtaining a cell; and   b. genetically modifying the cell by inserting at least one sequence encoding a transgene within a safe harbor locus, wherein the safe harbor locus is at any one or more of an sgRNA target loci selected from: chr10:33130000-33140000, chr10:72290000-72300000, chr11:128340000-128350000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000, chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, chr9:7970000-7980000, APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.