Multiplex Genome Editing With Genomic Barcodes for Engineered Cells
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Solution Overview
Problem
Existing methods for genome editing using RNA-guided nucleases are limited by inefficient homologous recombination repair (HR) in metazoan cells, restricting the production of variant libraries to pools and hindering the characterization of individual variants.
Innovation Solution
A method for multiplex production and validation of genetically engineered cells using RNA-guided nucleases and barcoding, which facilitates precise genome editing at desired target chromosomal loci through homology directed repair, integrating guide RNA and donor DNA sequences as genomic barcodes for easy identification and isolation of individual variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 system is used for genome editing in metazoan cells, then precise genome editing capability is achieved, but homologous recombination repair efficiency remains low (10-60% maximal efficiency)
Solution Approach 1:
The patent uses an RNA-guided nuclease complex (Cas9-gRNA) as an intermediary to deliver the editing function to the target locus. The gRNA acts as a mediator that guides the nuclease to the specific genomic location, enabling precise editing while the cell's own HR machinery performs the repair. This intermediary approach allows precise targeting without directly enhancing the HR repair mechanism itself.
Solution Approach 2:
The patent optimizes multiple parameters to improve HR efficiency: donor DNA length (100-150 nt), gRNA specificity-determining region (20 nt), donor DNA concentration, and timing of donor DNA addition. These parameter changes collectively enhance the HR repair efficiency from the baseline 10-60% to achieve near 100% editing efficiency in pooled variants.
2Productivity
If pooled variant libraries are produced, then high-throughput production is achieved, but individual variant characterization becomes impossible
Solution Approach 1:
The patent creates a copy of the guide RNA and donor DNA sequences and integrates them as a genomic barcode at a chromosomal locus separate from the target locus. This barcode copy serves as a permanent record of the variant identity that can be read later to identify and characterize individual variants from the pooled library.
Solution Approach 2:
The patent separates the target locus (where editing occurs) from the barcode locus (where identification information is stored). This segmentation allows the pooled library to be produced and screened based on phenotype, while the barcode at a separate locus enables subsequent identification and characterization of individual variants through sequencing.
3Speed
If non-homologous end joining is preferred in metazoan cells, then rapid DNA repair is achieved, but precise genetic changes are limited
Solution Approach 1:
The patent provides donor DNA in excess (100-150 nt length) to outcompete the NHEJ pathway. By providing abundant donor DNA with sufficient homology arms, the system biases repair toward the more precise HR pathway while still allowing the faster NHEJ pathway to operate on a smaller fraction of breaks. The excess donor DNA ensures that when HR does occur, precise editing is achieved.
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
Enables high-throughput multiplex genome editing with enhanced HR efficiency, allowing for precise genetic changes and efficient production of variant libraries, facilitating easy identification and validation of individual variants.
Implementation Method 1
a first nucleic acid sequence encoding a first guide RNA (gRNA) capable of hybridizing at a genomic target locus
Implementation Method 2
the RNA-guided nuclease in each cell forms a complex with the gRNA thereby forming a gRNA-RNA-guided nuclease complex
Implementation Method 3
allowing the gRNA-RNA-guided nuclease complex to modify the genomic target locus by integrating the donor polynucleotide into the genomic target locus
Data Source
AI summary
The present disclosure relates to multiplex production and phenotyping of genetically engineered cells using RNA-guided nucleases and genomic barcoding. In particular, high-throughput multiplex genome editing is achieved utilizing a system that facilitates precise genome editing at desired target chromosomal loci by homology directed repair. Integration of guide RNA and donor DNA sequences as a genomic barcode at a separate chromosomal locus allows identification, isolation, and massively-parallel validation of individual variants from a pool of transformants. Strains can be arrayed according to their precise genetic modifications, as specified by donor DNA incorporation in heterologous or native genes. The present disclosure further relates to a method of editing codons outside of canonical guide RNA recognition regions, which enables complete saturation mutagenesis of protein-coding genes, a marker-based internal cloning method, which removes background due to oligonucleotide synthesis errors and incomplete vector backbone cleavage, and a method of enhancing homology directed repair by active donor recruitment.