Method for constructing gene site-directed mutation

Abstract

The invention discloses a method for constructing gene site-directed mutation. The method comprises the following steps: determining a target sequence which is in a rat target genome sequence and can be identified by an artificial modified CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein) system; constructing a gRNA (guide ribonucleic acid) which can identify a CAS protein and guide the CAS protein to a target gene target sequence; introducing the gRNA sequence and a CAS protein coded deoxyribonucleic acid sequence or the gRNA sequence and an in-vitro expressed CAS protein mixture into a rat embryonic cell. According to the method, a homologous recombined targeting vector does not need to be constructed, ES (embryonic stem) targeting cell screening is not needed, a mutant has a high proportion, the operation is convenient, simultaneous multi-site knockout can be realized, the experiment cost can be greatly reduced, and the experiment cycle can be shortened.

Description

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AI Technical Summary

Benefits of technology

The technical effect of this patented method lies within three aspects - faster learning speed, lower resource consumption, easier handling compared to existing techniques such as phage display and random insertional clone rearrangement. Additionally, the use of direct injection without fusion cascade allows for quick creative cloning of wild ungulate models while maintaining their original functions like metabolism. Overall, these improvements make it safer and economically viable when studying rodents and mouse monoclonal antibodies against disease related proteins.

Problems solved by technology

Technologies aim towards improving the efficiency and versatility of modifying proteins involved in biological processes like metabolism, cellular signal processing, disease detection, etc., particularly when dealing with rare mutations present within these structures. Current approaches involve assembling multiple copies of cloned templates onto chromosomal elements, but they require extensive effort and may lead to instability issues. To overcome these challenges, we propose a method involving modulation and assembly techniques based upon molecular recognition functions generated by enzymatic domains associated with DNA recognition mechanisms.

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