Search

EP-4301868-B1 - METHODS AND COMPOSITIONS FOR COMBINATORIAL TARGETING OF THE CELL TRANSCRIPTOME

EP4301868B1EP 4301868 B1EP4301868 B1EP 4301868B1EP-4301868-B1

Inventors

  • SANJANA, Neville, E.
  • WESSELS, Hans-Hermann
  • MENDEZ-MANCILLA, Alejandro
  • SATIJA, Rahul

Dates

Publication Date
20260506
Application Date
20220301

Claims (15)

  1. A nucleic acid comprising a CRISPR array comprising one or more crRNA sequences, each crRNA comprising a direct repeat sequence and a gRNA sequence, and a 5' direct repeat sequence linked to a barcode guide RNA (bcgRNA), the bcgRNA comprising from 5' to 3' (a) optionally, a PCR handle; (b) a barcode sequence; and (c) a reverse-transcription handle.
  2. The nucleic acid according to claim 1, wherein the CRISPR array comprises one, two, three, or more crRNA sequences, optionally wherein each of the one or more crRNA sequences comprises a gRNA that comprises an at least 20 nucleotide sequence that is complementary to a target RNA sequence.
  3. The nucleic acid according to claim 1 or 2, wherein each of the crRNA present in the CRISPR array has a different gRNA sequence.
  4. The nucleic acid according to claim 1 or 2, wherein two or more of the crRNA present in the CRISPR array comprise the same gRNA sequence.
  5. The nucleic acid according to any one of claims 1 to 3, wherein each of the crRNA sequences present in the CRISPR array is specific for a different region of a target transcript.
  6. The nucleic acid according to any one of claims 1 to 5, wherein the CRISPR array comprises crRNA sequences having guide RNA (gRNA) sequences that target multiple transcripts.
  7. The nucleic acid according to any one of claims 1 to 6, wherein the bcgRNA is downstream (3') of the one or more crRNA sequences of the CRISPR array.
  8. The nucleic acid according to any one of claims 1 to 6, wherein the bcgRNA is upstream (5') of the one or more crRNA sequences of the CRISPR array.
  9. The nucleic acid according to any one of claims 1 to 8, wherein (a) the direct repeat is capable of binding a CRISPR-Cas13 enzyme, optionally Cas13d; (b) the reverse-transcription handle comprises a polyA sequence, a CS1, or a CS2; (c) the barcode comprises 8 to 15 nucleotides; and/or (d) the CRISPR array further comprises a stabilizing RNA element at its 3' end, optionally wherein the stabilizing RNA element is a MALAT1, NEAT1 (MENB), spnpreQ 1 , ZIKV xrRNA1, mpknot, or evopreQ 1 element or the stabilizing RNA element comprises SEQ ID NO: 39, 40, 41, 42, 43, or 44.
  10. An expression cassette comprising a sequence encoding the nucleic acid according to any one of claims 1 to 9.
  11. A vector comprising the expression cassette according to claim 10, wherein the vector is a non-viral vector, optionally a plasmid, or a viral vector, optionally a retrovirus vector, a lentivirus vector, an adenovirus vector, or an adeno-associated virus vector.
  12. The vector according to claim 11, wherein the plasmid comprises a sequence encoding a CRISPR-Cas enzyme, optionally a Cas13 enzyme, a Cas12 enzyme, or a type VI CRISPR-Cas enzyme.
  13. A host cell comprising the nucleic acid according to any one of claims 1 to 9, or the expression cassette according to claim 10, and a CRISPR-Cas enzyme, optionally a Cas13 enzyme or a Cas12 enzyme.
  14. A method of introducing one or more gene perturbations in a single cell transcriptome, the method comprising culturing the host cell according to claim 13.
  15. A method of performing gene perturbation profiling, the method comprising: (a) obtaining the host cell according to claim 13, optionally labeling the cell with a fluorophore-conjugated antibody, and sorting the cell using flow cytometry; (b) isolating RNA from the cell; (c) performing reverse-transcription comprising contacting the RNA with a primer specific for the reverse-transcription handle; (d) identifying the barcode sequence, optionally by amplifying the barcode sequence using a primer specific for the PCR handle; and (e) detecting expression of one or more transcripts or gene products, optionally by using one or more of flow cytometric analysis, cell-hashing, single-cell sequencing analysis, single cell RNA sequencing (scRNA-seq), Perturb-seq, CROP-seq, CRISP-seq, ECCITE-seq, cellular indexing of transcriptomes and epitopes (CITE-seq), wherein the CRISPR-Cas enzyme introduces one or more perturbations in the cell transcriptome.

Description

STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under DP2HG010099, DP2HG009623-01, RM1HG011014-01, and R01CA218668 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Recent technological advances that couple pooled genetic perturbations with scRNA-seq or multimodal characterization (i.e. Perturb-Seq, CROP-seq, CRISP-seq, and ECCITE-seq) promise to transform our understanding of gene function. In particular, the ability to perform combinatorial perturbations represents an opportunity to decode complex regulatory networks, with pioneering work demonstrating the ability to identify epistasis and other genetic interactions. However, there are specific technical and analytical challenges associated with pooled single-cell screens which are exacerbated when considering combinatorial perturbations. For example, undetected or incorrectly assigned sgRNAs can affect up to 20% of cells, but this is compounded when multiple independent sgRNAs are introduced and independently detected in each cell. Moreover, perturbations introduced by Cas9 are not uniformly efficient, and a considerable fraction of targeted cells may exhibit no phenotypic effects of perturbation. Therefore, when performing two or more simultaneous perturbations, the fraction of cells where all perturbations are both successfully introduced and successfully detected can decrease dramatically. WO2018005691A1 discloses a genetic screening method using guide RNAs that contain a barcode and a poly(A) tail, which allows CRISPR-based genetic perturbations to be tracked at the single-cell level by RNA sequencing. The method enables identification of the effects of specific genetic modifications on gene expression in individual cells. Dixit, A., Parnas, O., Li, B., et al., Cell, 167(7):1853-1866.e17, 2016, describes the Perturb-seq technique, which combines pooled CRISPR-mediated gene perturbations with single-cell RNA sequencing. This approach assigns transcriptional profiles to individual genetic perturbations and allows analysis of genetic interactions in cell populations. WO2016182959A1 discloses methods for CRISPR/Cas9-mediated gene editing in stem cells, involving transient treatment of the cells with viability-enhancing compounds before and/or after delivery of CRISPR/Cas9 components. The document also describes the use of guide RNAs with 5' caps and 3' poly(A) tails to affect editing efficiency, cell viability, and engraftment. What is needed is improved compositions and methods for introducing multiple genetic perturbations in a cell. SUMMARY OF THE INVENTION The subject-matter for which protection is sought is defined by the attached claims; any embodiments described herein that are not encompassed by the claims are provided for illustrative purposes only. In one aspect, provided herein is a nucleic acid comprising a CRISPR array comprising one or more crRNA sequences, each crRNA comprising a direct repeat (DR) sequence and a gRNA sequence, and a 5' direct repeat (DR) sequence linked to a barcode guide RNA (bcgRNA), the bcgRNA comprising from 5' to 3' (a) a barcode sequence; and (b) a reverse-transcription handle. In certain embodiments, the bcgRNA comprises from 5' to 3' (a) a PCR handle; (b) a barcode sequence; and (c) a reverse-transcription handle. In certain embodiments, the CRISPR array comprises one, two, three, or more crRNA sequences. In certain embodiments, each of the one or more crRNA sequences comprise a gRNA that comprises an at least 20 nucleotide sequence that is complementary to a target RNA sequence. In certain embodiments, each of the one or more crRNA sequences comprise a gRNA that is a 23-nucleotide sequence that is complementary to a target RNA sequence. In certain embodiments, each of the crRNA present in the CRISPR array has a different gRNA sequence. In certain embodiments, two or more of the crRNA present in the CRISPR array comprise the same gRNA sequence. In certain embodiments, each of the crRNA sequences present in the CRISPR array is specific for a different region of a target transcript. In certain embodiments, the CRISPR array comprises crRNA sequences having guide RNA (gRNA) sequences that target multiple transcripts. In certain embodiments, the bcgRNA is downstream (3') of the one or more crRNA sequences of the CRISPR array. In certain embodiments, the bcgRNA is upstream (5') of the one or more crRNA sequences of the CRISPR array. In certain embodiments, the direct repeat is capable of binding a CRISPR-Cas13 enzyme, optionally Cas13d. In certain embodiments, the reverse-transcription handle comprises a polyA sequence, a CS1, or a CS2. In certain embodiments, the barcode comprises 8 to 15 nucleotides. In certain embodiments, CRISPR array further comprises a stabilizing RNA element at its 3' end. In certain embodiments, the stabilizing RNA element is a MALAT1, NEAT1 (MENß), spnpreQ1, ZIKV xrRNA1, mpknot, or evopreQ1 element. In an