while studying about gene editing techniques, I came to know about the term targeted mutagenesis.
it was explained that after cutting the DNA at specific site using engineered nuclease, if homologous DNA not provided it leads to NHEJ which in turn leads to random mutation at the cut site. this was called as targeted mutagenesis.
my doubt is whether targeted mutagenesis and site specific mutagenesis are same are different.
please quote some references
targeted mutagenesis can really mean any mutagenic approach that can be applied to a specific gene or region. There are many different approaches to do this and site specific cleavage to promote mutagenesis is one of them.
Site specific mutagenesis generally refers to oligonucleotide driven mutagenesis which is specifically targeted by the oligo and the specific mutation(s) defined by the oligo. So they are not really the same although one could argue that site specific mutagenesis is a subset of targeted.
Hello R. Mahalakshmi
Site-specific mutagenesis is a method to create specific, targeted changes in double stranded DNA causing a predetermined amino acid change. These substitution mutations can result in drastic changes in protein conformation and function. There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), which include
a. To study changes in protein activity that occur as a result of the DNA manipulation.
b. To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property.
c. To introduce or remove restriction endonuclease sites or tags.
On the other hand, targeted mutagenesis is mainly used in CRISPR/Cas9 technology which is a simple and efficient technology of gene editing that enables the modification of genetic information in a targeted manner. It has two vital components namely, a guide RNA (gRNA) that binds complementary to a target DNA sequence, and an endonuclease (Cas9) that causes a dsDNA break that requires repair. Cas9-induced site-specific dsDNA breaks induce endogenous cell repair mechanisms, which can be exploited to modify the DNA. The error-prone nonhomologous end-joining (NHEJ) can rapidly ligate DNA breaks; however, it may generate small insertions and deletions that can cause frameshift mutations. Alternatively, dsDNA breaks can be repaired by homology-directed repair (HDR), which can insert exogenous DNA and introduce precise genome editing. The NHEJ pathway is commonly used to create targeted mutations.
You may want to refer to the articles attached below for more information.
https://www.ncbi.nlm.nih.gov/books/NBK560519/#:~:text=Site%2Ddirected%20Mutagenesis,in%20protein%20conformation%20and%20function.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217109/
https://doi.org/10.1104/pp.15.00793
Best.
Hello Sir,
They are not the same. Site-directed mutagenesis is a specific type of targeted mutagenesis that focuses on introducing mutations at predefined sites in a gene or plasmid. Targeted mutagenesis, on the other hand, encompasses a broader range of techniques that enable the precise modification of genetic material at specific target sites, and it is not limited to single-nucleotide changes.
Thank you for the accurate and interesting views. I'm actually wondering the considerations one would make, when he/she chooses a target gene and vector design. More specifically the genetic consideration in replacing or adding a gene into mammalian cell types.
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