Multiplex RNA-Guided Genome Engineering via Pre-Assembled Ribonucleoprotein Complexes
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Solution Overview
Problem
Current methods for multiplex DNA modification in cells are inefficient and require multiple steps to achieve multiple genetic modifications, limiting the rate of homologous recombination and the ability to introduce exogenous donor nucleic acids into specific sites in DNA.
Innovation Solution
The use of guide RNAs to direct an enzyme, such as the Cas9 protein, to specific sites in DNA for cutting and subsequent insertion of exogenous donor nucleic acids through homologous recombination, allowing for repeated cycles of DNA modification to achieve multiple alterations in a single cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple exogenous nucleic acid insertions are accomplished by repeated steps of introducing RNA and exogenous nucleic acids into cells, then multiple genetic modifications can be achieved, but the process is time-consuming and reduces productivity
Solution Approach 1:
The patent applies preliminary action by pre-assembling ribonucleoprotein complexes containing the enzyme and multiple guide RNAs before introducing them into the cell. This pre-assembly allows multiple target sites to be targeted simultaneously in a single transformation step, eliminating the need for repeated sequential transformations and thereby increasing productivity while maintaining the ability to achieve multiple genetic modifications.
2Productivity
If a single step of introducing multiple RNAs and exogenous donor nucleic acids is used, then productivity increases, but the complexity of the transformation process increases
Solution Approach 1:
The patent applies segmentation by dividing the complex transformation process into distinct functional modules: (1) guide RNA molecules that specify target sequences, (2) ribonucleoprotein complexes that provide the catalytic activity, and (3) exogenous donor nucleic acids that provide the replacement sequences. This modular segmentation allows each component to be optimized independently and simplifies the overall process design while maintaining high productivity through simultaneous multiplex editing.
3Manufacturing precision
If guide RNAs are used to direct enzyme to specific DNA sites, then manufacturing precision of genetic modifications improves, but the difficulty of detecting and measuring successful modifications increases
Solution Approach 1:
The patent applies the color changes principle by utilizing selectable marker genes that confer detectable phenotypes, such as antibiotic resistance or fluorescent protein expression, in the exogenous donor nucleic acids. These phenotypic changes serve as visual or selectable indicators that allow straightforward identification and selection of cells that have successfully incorporated the donor DNA at the targeted loci, thereby reducing the difficulty of detection despite the high precision requirements of the guide RNA-directed editing.
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
This approach significantly increases the rate of homologous recombination, enabling efficient multiplex genetic modification of cells by enabling multiple insertions of exogenous DNA at specific sites, including negative selection against cells that do not incorporate donor DNA, thereby identifying cells with high recombination frequency.
Implementation Method 1
guide RNAs to direct an enzyme having nuclease activity expressed by the cell, such as a DNA binding protein having nuclease activity, to a target location on the DNA
Implementation Method 2
the enzyme cuts the DNA and an exogenous donor nucleic acid is inserted into the DNA
Implementation Method 3
an exogenous donor nucleic acid is inserted into the DNA, such as by homologous recombination
Data Source
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AI summary
Methods of multiplex genome engineering in cells using Cas9 is provided which includes a cycle of steps of introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells