Cas9 Guide RNA Genome Editing for hiPSC DNA Insertion
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome editing tools, such as TALENs, face challenges in efficiently targeting specific DNA sequences due to repetitive sequences that complicate DNA construct synthesis and impair lentiviral gene delivery, while existing assays for evaluating NHEJ and HDR have limited sensitivity and accuracy, particularly in human induced pluripotent stem cells (hiPSCs).
Innovation Solution
The use of modified TALENs lacking repeat sequences of specific lengths, combined with guide RNAs, to specifically cleave target DNA and facilitate nonhomologous end joining or homologous recombination, along with the use of RNA-guided DNA binding proteins like Cas9 for precise DNA modification, enabling multiplex modifications and scarless genetic alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If TALENs with repeat sequences are used for genome editing, then DNA binding specificity is achieved, but DNA construct synthesis becomes complicated and lentiviral gene delivery is impaired
Solution Approach 1:
The patent extracts and removes the repetitive DNA sequences from the TALEN construct while retaining the essential DNA-binding functionality through alternative design elements, thereby simplifying DNA construct synthesis and enabling lentiviral delivery without compromising target specificity
Solution Approach 2:
The patent segments the TALEN construct into modular functional domains, separating the DNA-binding function from the repetitive sequences that cause synthesis complications, allowing independent optimization of each module for both specificity and manufacturability
2Reliability
If TALENs with repeat sequences are used for genome editing, then DNA binding specificity is achieved, but lentiviral gene delivery is impaired
Solution Approach 1:
The patent removes the repetitive sequences that are incompatible with lentiviral packaging constraints, retaining only the essential functional elements required for DNA binding, thereby enabling successful lentiviral delivery while maintaining target specificity
Solution Approach 2:
The patent changes the physical parameters of the TALEN construct by reducing its size and eliminating repetitive elements, making it compatible with the size constraints and delivery requirements of lentiviral vectors
3Productivity
If conventional NHEJ and HDR assays are used for evaluating genome editing, then editing frequencies can be assessed, but sensitivity and accuracy are limited
Solution Approach 1:
The patent introduces an intermediary reporter system that amplifies the signal from editing events, using a detectable intermediate marker to enhance the sensitivity and accuracy of measuring low-frequency editing events in hiPSCs
Solution Approach 2:
The patent nests a reporter gene within the target locus, creating a nested structure where the reporter serves as an internal control and amplification mechanism, allowing highly sensitive detection of editing events while maintaining physiological relevance
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
Enhances genome editing efficiency in hiPSCs, achieving high frequencies of targeted DNA alterations and insertions, with improved sensitivity and accuracy in assessing editing rates, and allowing for reversible genetic modifications without leaving residual vectors.
Implementation Method 1
the TALEN cleaves the target DNA and the cell undergoes nonhomologous end joining to produce altered DNA in the cell
Implementation Method 2
a guide RNA and a DNA binding protein, such as Cas9, that specifically cleave a target DNA sequence
Data Source
AI summary
Methods are provided for altering target DNA in a cell genetically modified to express a Cas 9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner. Methods include introducing into the cell a first foreign nucleic acid encoding a donor nucleic acid sequence, introducing into the cell from media surrounding the cell the guide RNA complementary to the target DNA and which guides the Cas 9 enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, wherein the donor nucleic acid sequence is expressed, wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell.