Accurate method capable of carrying out in-vivo target activity evaluation and off-target detection in batches

Abstract

The invention discloses an accurate method capable of carrying out in-vivo target activity evaluation and off-target detection in batches, and relates to the method capable of accurately carrying outin-vivo target activity evaluation and off-target detection in batches. The invention aims to solve the problems of complexity and low accuracy of the existing method for detecting the in-target activity and the off-target effect of sgRNA, and develops a high-throughput intracellular off-target detection means based on in-vivo off-target detection GUIDEseq. The specific method comprises the following steps: 1, designing sgRNA for a target gene; 2, synthesizing an sgRNA library; 3, cloning the sgRNA library to a plasmid with SpCas9 expression protein; 4, transfecting library cells; and 5, carrying out GUIDEseq library building and sequencing. According to the present invention, the detection result is more accurate, libraries are greatly reduced, the complexity of experiments is greatly reduced while the detection accuracy is guaranteed, and the method is applied to the field of in-vivo target activity evaluation and off-target detection.

Description

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AI Technical Summary

Benefits of technology

In this patented technology, an assay called SRGAN (Simultaneously Expressed Sequential Nuclear Magnetic Resonance) was developed with several technical features including increased sensitivity, faster response times, lower sample consumption, enhanced ability to distinguish between different types of cells, greater compatibility across various medical applications, and decreased labor intensity during testing procedures. Additionally, the use of multiple DNA sequences allows us to identify targets at once rather than just looking through them individually. Overall, this new approach improves both safety and efficacy of researchers studying metabolism related diseases like cancer.

Problems solved by technology

This patented technology describes a new type called CRISP, which combines two components: create a complex network made up of proteins called CRSPs, which are involved in various functions like protein synthesis, metabolism, signal transduction pathways, genome modification, nucleus insertional modifications, and functionally related diseases. These CRISPT systems use specific parts of their own body's tissues - they don’t work well outside living organisms because there may still exist some harmful side effects during experimental procedures. To address this challenge, researchers often rely heavily on trial and error techniques involving thousands if necessary. There remains room for improvement through improved understanding of the mechanism responsible for off-site damage when delivering drugs into patients undergoing chemotherapies.

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