Barcoding sequences for identification of gene expression

Abstract

Gene expression can be identified by analyzing a DNA sequence. The DNA sequence can include a barcode sequence that corresponds to a particular gene. The barcode sequence can be produced during the expression of a gene by first adding a Homologous Directed Repair (HDR) template including the barcode sequence into the DNA sequence of the gene and then splicing the barcode sequence out of an RNA precursor during the expression of the gene. As the barcode sequence is made available from the RNA precursor, it can be added to the DNA strand using HDR. The resulting DNA strand can be sequenced and the sequence data can be analyzed to identify the barcode sequence within the DNA sequence, which provides an indicator of the expression of the gene in DNA rather than RNA.

1 . A method for identifying expression of a gene, the method comprising:
 producing, in response to expression of the gene, a gene product including at least a first splicing region and a barcode sequence, the first splicing region including a first sequence of nucleotides that is recognized by an enzyme to produce a cut in the first splicing region and the barcode sequence including a sequence of nucleotides that uniquely identifies the gene;   splicing, using the enzyme, a homology directed repair (HDR) template from the gene product by cutting at least the first splicing region, the HDR template including a sequence of nucleotides that includes a portion of the first splicing region and the barcode sequence;   inserting the HDR template into a double stranded polynucleotide using HDR;   sequencing the double stranded polynucleotide to produce sequencing data; and   determining that the gene has been expressed based at least partly on identifying the sequence of nucleotides of the barcode sequence in the sequencing data.   
     
     
         2 . The method of  claim 1 , wherein:
 the gene product includes a second splicing region;   a portion of the first splicing region is homologous to a first portion of a target site in the double stranded polynucleotide; and   a portion of the second splicing region is homologous to a second portion of the target site in the double stranded polynucleotide.   
     
     
         3 . The method of  claim 1 , wherein the barcode sequence is located in a 3′ untranslated region of the gene. 
     
     
         4 . The method of  claim 1 , wherein the enzyme is a spliceosome, and the method further comprises designing the gene product such that the first splicing region includes a sequence of nucleotides recognized by the spliceosome and the HDR template remains viable to perform HDR with the double stranded polynucleotide for a period of time. 
     
     
         5 . The method of  claim 1 , further comprising:
 modifying the gene by inserting, via HDR, a first HDR template that adds the first splicing region and the barcode sequence to the gene.   
     
     
         6 . The method of  claim 1 , further comprising:
 generating data indicating a plurality of barcode sequences, wherein the gene is one of a plurality of genes; and   uniquely associating individual genes of the plurality of genes with a one of the plurality of barcode sequences such that each barcode sequence of the plurality of barcode sequences corresponds to only one of the individual genes.   
     
     
         7 . The method of  claim 1 , wherein the gene product is an RNA precursor that is a single stranded polynucleotide which includes:
 a first sequence that corresponds to the first splicing region;   a second sequence that corresponds to the barcode sequence;   a 3′ untranslated region (UTR) and a 5′ UTR; and   a coding region that includes an intron and an exon.   
     
     
         8 . The method of  claim 7 , wherein the RNA precursor includes a gene expression region that comprises the barcode sequence. 
     
     
         9 . The method of  claim 8 , wherein the splicing of the gene expression region from gene product produces the HDR template. 
     
     
         10 . A system for identifying expression of a gene, the system comprising:
 a gene, that when expressed, produces a gene product, wherein the gene product includes at least a first splicing region and a barcode sequence, the first splicing region including a first sequence of nucleotides that is recognized by a first enzyme to produce a cut in the first splicing region and the barcode sequence including a sequence of nucleotides that uniquely identifies the gene;   the first enzyme configured to splice a homology directed repair (HDR) template from the gene product by cutting at least the first splicing region, wherein the HDR template includes a sequence of nucleotides that includes a portion of the first splicing region and the barcode sequence;   a double stranded polynucleotide including a target site, wherein a first subsequence of the target site hybridizes to a first sequence of the HDR template and a second subsequence of the target site hybridizes to a second sequence of the HDR template such that the double stranded polynucleotide is configured to incorporate the HDR template using HDR; and   a second enzyme configured to create a double strand break in the double stranded polynucleotide at a cut site in the target site.   
     
     
         11 . The system of  claim 10 , wherein the first enzyme is a spliceosome and the second enzyme is a restriction enzyme, a homing endonuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, CRISPR/Cas, or NgAgo. 
     
     
         12 . The system of  claim 10 , wherein the double stranded polynucleotide is at least one of genomic DNA, artificial DNA, circular DNA, or linear DNA. 
     
     
         13 . The system of  claim 10 , wherein the barcode sequence is located in a 3′ untranslated region of the gene. 
     
     
         14 . The system of  claim 10 , wherein:
 the gene product includes a second splicing region;   the barcode sequence is located between the first splicing region and the second splicing region;   a portion of the first splicing region is homologous to a first portion) of a target site; and   a portion of the second splicing region is homologous to a second portion) of the target site.   
     
     
         15 . The system of  claim 10 , wherein the gene product is an RNA precursor that is a single stranded polynucleotide which includes:
 a first sequence that corresponds to the first splicing region;   a second sequence that corresponds to the barcode sequence;   a 3′ untranslated region (UTR) and a 5′ UTR; and   a coding region that includes an intron and an exon.   
     
     
         16 . The system of  claim 10 , wherein the double stranded polynucleotide includes a second target site, the second target site configured to incorporate via HDR a second HDR template generated from a second gene product expressed by a second gene, the second HDR template including a second barcode sequence uniquely identifies the second gene. 
     
     
         17 . The system of  claim 10 , wherein the gene includes a target site;
 a third enzyme configured to create a double strand break in the gene at a cut site in the target site; and   a first HDR template configured to add the first splicing region and the barcode sequence to the gene by HDR.   
     
     
         18 . The system of  claim 10 , further comprising a polynucleotide sequencer configured to sequence the double stranded polynucleotide and produce sequencing data. 
     
     
         19 . The system of  claim 18 , further comprising a digital computer comprising a sequence data analyzer configure to identify the barcode sequence in the sequencing data. 
     
     
         20 . The system of  claim 19 , wherein:
 the gene is configured to be expressed in response to a signal that occurs at a particular time; and   the sequence data analyzer is further configured to analyze the sequencing data and determine a period of time that the gene was expressed based at least partly on the presence of the barcode sequence in the sequencing data.