Encoding of dna vector identity via iterative hybridization detection of a barcode transcript

Abstract

Embodiments disclosed herein are directed to a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH—in conjunction with a large array of genetic perturbations applied as a pool in a single test volume.

1 - 36 . (canceled) 
     
     
         37 . A method for screening cells for comprising:
 culturing a cell or population of cells in one or more discrete volumes;   a) introducing one or more vectors into the cell or cell population, each vector comprising nucleic acid sequences encoding   i) one or more optical barcodes, each optical barcode comprises a set of ordered segments, each segment comprising a nucleic acid base sequence from a set of possible nucleic acid bases or sequences for that particular segment; and   ii) one or more genetic perturbations;   thereby introducing the one or more optical barcodes and the one or more genetic perturbations into the cell;   b) incubating the cells to allow for expression of RNA comprising the one or more optical barcodes; and   c) detecting the one or more optical barcodes in the RNA to identify the one or more genetic perturbations present in the cell or cell populations.   
     
     
         38 . The method of  claim 37 , further comprising determining an observed phenotype for each cell or cell population by capturing a microscopic image of the cell or cell population; and correlating the observed phenotype to the one or more detected optical barcodes. 
     
     
         39 . The method of  claim 37 , wherein detecting the one or more optical barcodes comprises:
 delivering a probe set to the cell or cell population, each probe in the probe set comprising a sequence that hybridizes to one of the possible nucleic acid sequences at the first segment of the optical barcode on the RNA, wherein different probe sequences are labeled with different optically detectable labels such that each nucleic acid sequence at the first segment of the optical barcode is labeled with a different optically detectable label;   determining the nucleotide sequence at the first segment of each barcode by detecting the optically detectable labels; and   repeating the delivering and determining steps for each segment in the barcode to detect all remaining segments in the optical barcode, wherein the order in which the probes in the probe set bind to the segments of the optical barcode identify each optical barcode and thereby identify the one or more genetic perturbations introduced into each cell or cell population.   
     
     
         40 . The method of  claim 38 , further comprising generating a cDNA copy of the RNA prior to detecting the optical barcode. 
     
     
         41 . The method of  claim 40 , further comprising amplifying the generated cDNA copy prior to detecting the optical barcode. 
     
     
         42 . The method of  claim 37 , wherein detecting the optical barcode comprises detecting the nucleic acid sequence of the ordered segments in the optical barcode using an in situ sequencing method. 
     
     
         43 . The method of  claim 42 , wherein the in situ sequencing method is fluorescent in situ RNA sequencing (FISSEQ) or in situ mRNA-seq. 
     
     
         44 . The method of  claim 37 , wherein detecting the one or more optical barcodes comprises:
 delivering an intermediate probe set to the cell or cell population, each probe in the probe set comprising a sequence that hybridizes to one of the possible nucleic acid sequences at the first segment of the optical barcode on the RNA;   delivering a second probe set that comprises probes that bind to the intermediate probes, the probes in the second probe set comprising an optically detectable label;   determining the nucleotide sequence at the first segment of each barcode by detecting the optically detectable labels; and   repeating the delivering and determining steps for each segment in the barcode to detect all remaining segments in the optical barcode, wherein the order in which the probes in the probe set bind to the segments of the optical barcode identify each optical barcode and thereby identify the one or more genetic perturbations introduced into each cell or cell population.   
     
     
         45 . The method of  claim 44 , wherein the intermediate probes are branched probes that facilitate the binding of multiple probes from the second probe set to a single intermediate probe. 
     
     
         46 . The method of  claim 37 , wherein the RNA comprising the barcode further comprises a localization signal localizing the RNA comprising the barcode to a specific location within the cell. 
     
     
         47 . The method of  claim 46 , wherein the cell localization signal is a nuclear localization signal. 
     
     
         48 . The method of  claim 37 , wherein the vector further comprises nucleic acid sequences further encoding the site-specific nuclease. 
     
     
         49 . The method of  claim 48 , wherein the genetic perturbation is a guide RNA. 
     
     
         50 . The method of  claim 37 , wherein the vector encodes guide RNA and the site-specific nuclease is an RNA-guided DNA endonuclease. 
     
     
         51 . The method of claim  5050 , wherein the RNA-guided DNA endonuclease is dCas9. 
     
     
         52 . The method of  claim 51 , wherein the dCas9 is fused to a second domain. 
     
     
         53 . The method of  claim 52 , wherein the second domain is a nickase. 
     
     
         54 . The method of  claim 53 , wherein the nickase is Fok1. 
     
     
         55 . The method of  claim 52 , wherein the second domain is selected from a transcriptional activator, or a transcriptional repressor, a recombinase, a transposase, a DNA methyltransferase or a histone methyltransferase. 
     
     
         56 . The method of  claim 50 , further comprising generating, prior to the introducing, a set of gateway vectors, the gateway vector comprising a guide RNA and corresponding optical barcode assigned to each guide RNA such that the guide RNA and optical barcode are adjacent to one another; and identifying each guide RNA and optical barcode pair using short-read DNA sequencing. 
     
     
         57 . The method of  claim 37 , wherein the optical barcode further comprises a unique molecular identifier (UMI), and each guide RNA sequence comprises a barcode-UMI specific homology sequence to facilitate one to one cloning of guide RNAs to corresponding optical barcodes. 
     
     
         58 . The method of  claim 37 , wherein the site-specific nuclease is a zinc-finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). 
     
     
         59 . The method of  claim 37 , wherein the one or more genetic perturbations comprises one or more nucleotide insertions, deletions, or substitutions. 
     
     
         60 . The method of  claim 37 , wherein the RNA comprising the barcode further comprises a premature termination signal to prevent translation of the RNA comprising the barcode. 
     
     
         61 . The method of  claim 37 , wherein each nucleic acid sequence at each segment in the barcode is between approximately 100 to approximately 200 nucleotides. 
     
     
         62 . The method of  claim 37 , wherein the barcode comprises 3 to 8 segments. 
     
     
         63 . The method of  claim 39 , wherein each probe set comprises 3, 4, or 5 distinct optically detectable labels. 
     
     
         64 . The method of  claim 37 , wherein the optically detectable label is a fluorophore. 
     
     
         65 . The method of  claim 37 , wherein the optically detectable label is a quantum dot. 
     
     
         66 . The method of  claim 37 , wherein the optically detectable label is an object of a particular size, shape, color, or combination thereof. 
     
     
         67 . The method of  claim 37 , further comprising sequencing the one or more vectors to identify the sequence defining the barcode associated with the one or more genetic perturbations. 
     
     
         68 . The method of  claim 37 , wherein the optical barcode further comprises a unique molecular identifier (UMI). 
     
     
         69 . The method of  claim 68 , further comprising sequencing the UMI and optical barcode to identify the UMI assigned to each optical barcode thereby allowing short sequencing of the UMI to identify the optical barcode associated with each vector and genetic perturbation or guide RNA encoded by that vector. 
     
     
         70 . The method of  claim 37 , further comprising selecting cells with increased RNA expression from the one or more vectors introduced into the cells, wherein the one or more vectors further encode a degradation domain fused to an antibiotic resistance protein such that rapid degradation of the expressed antibiotic resistance protein can be induced such that only cells with increased RNA expression are selected in the presence of an antibiotic. 
     
     
         71 . The method of  claim 37 , wherein the one or more genetic perturbations is an RNAi.