Endonuclease-barcoding

Abstract

The instant invention relates to a method for labeling endonuclease-treated cells, comprising the steps of a) providing cells or a composition comprising cells; b) bringing into contact said cells or said composition with: —at least one endonuclease suitable for targeting a genomic region of interest in said cells, or a vector suitable for expressing said endonuclease in said cells; —at least one first nucleic acid suitable for introducing one or more silent mutation(s) in said genomic region by homology-directed repair (HDR), and optionally one or more non-silent mutation(s); and —at least one second nucleic acid suitable for introducing one or more silent mutation(s) in said genomic region by homology-directed repair (HDR), but distinct from the silent mutations of the first nucleic acid; thereby labeling endonuclease-treated cells.

1 . A composition comprising at least:
 (i) a first population of endonuclease-treated cells, comprising one or more silent mutation(s) in a genomic region of interest of said cells; and   (ii) a second population of endonuclease-treated cells, comprising one or more silent mutation(s) in said genomic region, but distinct from the silent mutations of the first population;   wherein the silent mutation(s) is/are introduced in said cells by homology-directed repair (HDR) after treatment with a same endonuclease targeting said genomic region of interest.   
     
     
         2 . The composition of  claim 1 , wherein said first and second population of endonuclease-treated cells are obtained after bringing into contact cells with
 (i) at least one endonuclease suitable for targeting a genomic region of interest in said cells, or vector suitable for expressing said endonuclease; and   (ii) at least one nucleic acid suitable for introducing said silent mutation(s) in said genomic region of interest by HDR.   
     
     
         3 . The composition of  claim 1 , wherein said first and/or second population further comprise non-silent mutation(s) in said genomic region of interest. 
     
     
         4 . The composition of  claim 1 , wherein:
 (i) said first population comprises non-silent mutations but not the second population; or   (ii) said first and second populations both comprise non-silent mutation(s), said non-silent mutation(s) being the same or different, and preferably different.   
     
     
         5 . A kit for detecting a population of endonuclease-treated cells comprising:
 a first part comprising a first population of endonuclease-treated cells as defined in  claim 1 ; and   a second part comprising a second population of endonuclease-treated cells as defined in  claim 1 .   
     
     
         6 . A kit for labeling Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system-treated cells, comprising:
 (i) a first part comprising at least one CRISPR-Cas system comprising:   a) a first regulatory element operable in a cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target genomic region of interest of said cell, and   b) a second regulatory element operable in said cell operably linked to a nucleotide sequence encoding a Cas endonuclease, wherein components (a) and (b) are located on same or different vectors of the system; and   (ii) a second part comprising at least one nucleic acid suitable for introducing silent mutation(s) in the genomic region of interest of said cell by homology-directed repair (HDR), and optionally one or more non-silent mutation(s) in said genomic region of interest.   
     
     
         7 . The kit according to  claim 5 , comprising:
 at least one first nucleic acid suitable for introducing silent mutation(s) in said genomic region of interest by homology-directed repair (HDR), and optionally one or more non-silent mutation(s); and   at least one second nucleic acid suitable for introducing silent mutation(s) in said genomic region of interest by homology-directed repair (HDR), but distinct from the silent mutations of the first nucleic acid.   
     
     
         8 . The kit according to  claim 6 , comprising:
 at least one first nucleic acid suitable for introducing silent mutation(s) in said genomic region of interest by homology-directed repair (HDR), and optionally one or more non-silent mutation(s); and   at least one second nucleic acid suitable for introducing silent mutation(s) in said genomic region of interest by homology-directed repair (HDR), but distinct from the silent mutations of the first nucleic acid.   
     
     
         9 . The composition according to  claim 1 , wherein the endonuclease is selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease, an Argonaute nuclease (Ago), a mega nuclease (MN), a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN). 
     
     
         10 . The composition according to  claim 9 , wherein the endonuclease is a Type II-Cas9 endonuclease. 
     
     
         11 . The kit according  claim 5 , wherein the endonuclease is selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease, an Argonaute nuclease (Ago), a mega nuclease (MN), a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN). 
     
     
         12 . The kit according to  claim 11 , wherein the endonuclease is a Type II-Cas9 endonuclease. 
     
     
         13 . The kit according  claim 6 , wherein the endonuclease is selected from the group consisting of: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease, an Argonaute nuclease (Ago), a mega nuclease (MN), a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN). 
     
     
         14 . The kit according to  claim 13 , wherein the endonuclease is a Type II-Cas9 endonuclease. 
     
     
         15 . The composition according to  claim 1 , wherein the silent mutation(s) are within an open reading frame (ORF) or exon of a gene coding for a protein of interest. 
     
     
         16 . The composition according to  claim 1 , wherein said cell(s) is/are eukaryotic cell(s). 
     
     
         17 . The kit according to  claim 5 , wherein said cell(s) is/are eukaryotic cell(s). 
     
     
         18 . The kit according to  claim 6 , wherein said cell(s) is/are eukaryotic cell(s).