High-Throughput C-Terminal Gene Tagging With Orthogonal Cas Nucleases
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Solution Overview
Problem
Existing methods for gene tagging in mammalian cells are labor-intensive and can perturb protein function due to tags being inserted into the middle of the protein, limiting high-throughput and scalability.
Innovation Solution
The High-throughput Insertion of Tags Across the Genome (HITAG) system uses orthogonal Cas nucleases with non-homologous end joining to insert protein tags into the C-terminus of target genes, enabling scalable and efficient gene tagging in mammalian cells, allowing hundreds of genes to be tagged within a similar time frame.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If homologous recombination is used to insert tags at the C-terminus of target genes, then protein function is preserved, but the process involves significant labor for each line generated
Solution Approach 1:
The system divides the tagging process into two independent stages: (1) generating a cell pool with integrated gRNAs using high-throughput transfection, and (2) performing CRISPR-mediated tagging with donor plasmids. This segmentation allows parallel processing of multiple genes simultaneously, dramatically increasing productivity while maintaining the reliability of C-terminal tagging.
Solution Approach 2:
The system performs preliminary integration of gRNA encoding sequences into the genome before the actual tagging step. By pre-establishing a cell pool where each cell contains a specific gRNA, the system eliminates the need for simultaneous delivery of multiple gRNAs and donor plasmids, thereby simplifying the process and enabling high-throughput operation without compromising tagging accuracy.
2Productivity
If non-homologous end joining is used to insert synthetic exons containing protein tags into the introns of target genes, then tagging throughput is improved, but protein function is perturbed due to tag insertion into the middle of the protein
Solution Approach 1:
Instead of inserting tags into introns (as in previous NHEJ-based methods), the system inverts the approach by using NHEJ to create double-strand breaks at the C-terminus of target genes and then inserting donor plasmids that encode C-terminal tags. This inversion maintains the productivity advantage of NHEJ while restoring the reliability of C-terminal tagging by placing tags at the correct location.
Solution Approach 2:
The system introduces donor plasmids as intermediaries that contain both the C-terminal tag sequence and a selectable marker. These plasmids serve as mediators between the CRISPR-Cas9 system and the target gene, enabling precise C-terminal tagging through NHEJ while providing a mechanism for selection and verification of successful tagging events.
3Measurement precision
If traditional CRISPR methods are used for gene tagging, then specificity is achieved, but the process is labor-intensive and not scalable to library scales
Solution Approach 1:
The system merges multiple functions into a single integrated workflow: (1) high-throughput transfection of gRNA encoding sequences, (2) CRISPR-mediated genome editing, and (3) selectable marker-based enrichment. By combining these functions into one cohesive system, the method achieves both high specificity (through CRISPR guidance) and high productivity (through parallel processing and selection), enabling scaling to library levels.
Solution Approach 2:
The system employs selectable markers (e.g., antibiotic resistance genes) that enable cells with successful tagging events to self-select and proliferate under selective conditions. This self-service mechanism automates the enrichment of correctly tagged cells, eliminating the need for manual screening and verification, thereby dramatically increasing productivity while maintaining specificity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
HITAG achieves over 70% perfect fusion of tags with target genes without additional bases, facilitating efficient protein function interrogation and dynamics, and supports applications like protein localization and interaction network mapping.
Implementation Method 1
a first RNA-guided endonuclease, or a nucleic acid encoding thereof, configured to bind to the first guide RNA; and a second RNA-guided endonuclease, or a nucleic acid encoding thereof, configured to bind to the plurality of second guide RNAs
Implementation Method 2
uses orthogonal Cas nucleases with non-homologous end joining to insert protein tags into the C-terminus of target genes
Data Source
AI summary
The present disclosure provides systems and methods for high throughput genetic manipulation. Particularly, systems and methods are provided for scalable gene insertions in mammalian cells, the systems and methods comprise a donor nucleic acid comprising a cargo sequence encoding one or more selectable markers; a first guide RNA complementary to at least a portion of the donor nucleic acid; a plurality of second guide RNAs each of which is complementary to at least a portion of one of a plurality of target nucleic acids; a first RNA-guided endonuclease configured to bind to the first guide RNA; a second RNA-guided endonuclease configured to bind to the plurality of second guide RNAs; or one or more nucleic acids encoding thereof.