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Multiplex production and barcoding of genetically engineered cells

A genetic engineering, barcoding technology, applied in other directions of inserting foreign genetic material, genetic engineering, biochemical equipment and methods, etc., can solve problems such as limiting the phenotypic selection of characterizing individual variants

Pending Publication Date: 2020-06-26
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, generation of variant libraries has been limited to pools, which greatly limits options for characterizing individual variant phenotypes

Method used

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  • Multiplex production and barcoding of genetically engineered cells
  • Multiplex production and barcoding of genetically engineered cells
  • Multiplex production and barcoding of genetically engineered cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200]Example 1 describes the use of the yeast, Saccharomyces cerevisiae, for this purpose. Saccharomyces cerevisiae exists in diploid and haploid forms. Conjugation only occurs between yeast haploid forms of different conjugation types, which can be a or alpha conjugation types. The allele of the MAT locus (MATa or MATα) determines the mating type. Diploid cells were generated from crossing MATa and MATα yeast strains. Thus, haploid genetically modified yeast cells containing a gRNA-donor polynucleotide cassette can be hybridized with haploid barcoded yeast cells to generate diploid yeast cells, including gRNA-donor polynucleotide cassettes on different nucleic acids and barcode sequence. For example, a genetically modified yeast cell of strain MATα can be mated with a barcoded yeast cell of strain MATa. Alternatively, genetically modified yeast cells of strain MATa can be mated with barcoded yeast cells of strain MATa.

[0201] The gRNA-donor polynucleotide cassette is ...

Embodiment 2

[0341] Example 2. Genome Editing Using the Cpf1-Donor System Produces Efficient Editing

[0342] When the Cpf1 guide-donor system was used in a similar manner as described in Example 1, the Cpf1 guide-donor system caused efficient (>99%) editing and ~10-fold improvement with Cpf1 editing, with a degree of donor recruitment similar to Cas9.

[0343] Figure 14A and 14B provide data. Figure 14A Cell colonies showing pre-expression of Cpf1 were transformed with a Cpf1 guide-donor plasmid targeting the ADE2 gene (the guide has a Cpf1 scaffold). Donor DNA encodes a mutation that causes a frameshift. Figure 14B Shows % red colonies (ratio of red:white colonies) when Cpf1 guide-donor and non-editing plasmids were mixed at a ratio of 17:3 and transformed into Cpf1-expressing cells, without (left) or with (right) LexA-FHA .

Embodiment 3

[0344] Example 3. Plasmid Spike-In experiment proves that LexA-FHA and linearized vector improve HDR efficiency and editing survival.

[0345] ADE2ORF-editing plasmids were mixed at 85% (17:3) with non-editing plasmids and transformed into Cas9-carrying ( Figure 16 , top panel) or Cas9 and LexA-FHA ( Figure 16 , bottom panels) strains. Using the same strain for each transformation made direct comparison of total colonies per row feasible.

[0346] Figure 16 provide data. The y-axis indicates the total number of colonies observed in each transformation, while the x-axis indicates the percentage of colonies in red, which represents the survival of the process of editing ADE2. The shape of each spot corresponds to the restriction enzyme used to linearize the plasmid outside the transformation precursor. Five different columns correspond to different forms of spike-in mixtures. The first number corresponds to the number of genomic loci cleaved by the ADE2 editing plasmid ...

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Abstract

The present disclosure relates to the multiplex production and phenotyping of genetically engineered cells by using RNA-guided nucleases and genomic barcoding. In particular, the high-throughput multiplex genome editing is achieved by utilizing a system that facilitates the precise genome editing at desired target chromosomal loci by homology directed repair. The integration of guide RNA and donorDNA 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 canbe 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 the 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 the active donor recruitment.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 559,493, filed September 15, 2017, which is incorporated herein by reference in its entirety. [0003] Statement Regarding Federally Sponsored Research or Development [0004] This invention was made with government support under contract HG000205 awarded by the National Institutes of Health and contract 70NANB15H268 awarded by the National Institute of Standards and Technology. The government has certain rights in inventions. technical field [0005] The present disclosure relates generally to the field of genome engineering using RNA-guided nucleases. In particular, the present disclosure relates to compositions and methods for high-throughput generation and validation of genetically engineered cells using RNA-guided nucleases and barcode diversity. [0006] Background of the invention [0007] The advent of programmable genome editing via the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/85C12N15/90
CPCC12N15/81C12N15/85C12N15/907C40B30/06C12N15/102C12N2310/20C12N15/11C12N2320/10C12N2330/51C40B40/06C12Q2537/143C12N9/22C12N15/1082C12N15/113C12N2800/80
Inventor K·罗伊J·D·史密斯R·P·圣昂格L·M·施泰因梅茨J·E·哈伯
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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