Barcoded CRISPR Arrays for Detectable Multiplex Transcriptome Targeting
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Solution Overview
Problem
Existing methods for combinatorial genetic perturbations in single-cell screens face challenges such as undetected or incorrectly assigned sgRNAs, inefficient perturbation introduction, and reduced success rates when multiple perturbations are introduced, leading to significant fractions of cells not exhibiting phenotypic effects.
Innovation Solution
A CRISPR array comprising crRNA sequences and a barcode guide RNA (bcgRNA) with a reverse-transcription handle, enabling efficient and detectable combinatorial perturbations through a CRISPR-Cas13 system, allowing for simultaneous targeting of multiple genes and improved detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional methods (shRNA, RNAi, CRISPR/Cas9, CRISPRi) are used to target individual genes, then gene silencing or modulation can be achieved, but the complexity increases and off-target effects accumulate when attempting to target multiple genes simultaneously
Solution Approach 1:
The patent combines multiple gRNA expression cassettes into a single viral vector construct, allowing simultaneous delivery of multiple gRNAs (e.g., 2-12 gRNAs) to target multiple genes in one transduction event. This merging approach enables multiplexed gene targeting while maintaining a unified delivery system, thereby increasing versatility without proportionally increasing system complexity.
Solution Approach 2:
The invention creates a universal platform using a single viral vector (e.g., AAV, lentivirus) that can deliver variable numbers of gRNAs targeting different genes. The modular gRNA cassette design allows the same vector system to be adapted for targeting any combination of genes, providing multi-functionality and broad adaptability across different gene targets and experimental conditions.
2Measurement precision
If multiple individual viral vectors are used to target multiple genes, then each gene can be targeted specifically, but the number of transduction events and off-target effects increase
Solution Approach 1:
The patent merges multiple gRNA targeting functions into a single viral vector construct, enabling simultaneous delivery of multiple gRNAs in one transduction event. This single transduction approach maintains specific targeting of intended genes while eliminating the cumulative off-target effects associated with multiple sequential transductions, as the vector genome is integrated once rather than repeatedly.
Solution Approach 2:
The invention performs preliminary organization of multiple gRNAs into a single pre-assembled viral vector construct before delivery. This preliminary action ensures that all required gRNAs are delivered together in one transduction event, preventing the need for repeated transduction events that would increase off-target effects while maintaining precise targeting of multiple genes.
3Productivity
If traditional methods are used to screen for essential genes, then individual gene functions can be assessed, but the throughput and ability to identify multiple essential genes is limited
Solution Approach 1:
The patent merges the screening capability for multiple genes into a single experimental transduction event by incorporating multiple gRNAs targeting different genes within one viral vector. This enables simultaneous screening of multiple genes (e.g., 2-12 genes per vector) to identify essential genes, dramatically increasing throughput compared to traditional single-gene screening methods while maintaining accurate identification of essential genetic elements.
Data Source
Figure 1A~1B
Figure 1C~1E
Figure 2A
AI summary
Provided herein are compositions comprising a CRISPR array comprising one or more crRNA sequences and a 5' direct repeat (DR) sequence linked to a barcode guide RNA (bcgRNA), wherein the bcgRNA comprises from 5' to 3' (a) a barcode sequence, and (b) a reverse-transcription handle. Also provided are methods that comprise using the described CRISPR arrays to introduce one or perturbations in a single cell transcriptome.