Scientists have discovered two new methods capable of enhancing the efficiency of genetic engineering, and these new gene modification techniques could be huge when it comes to future research studies.
The two new methods are called the IsODN (long single-stranded oligodeoxynucleotide) and 2H2OP (two-hit two-oligo with plasmid) and are used in conjunction with CRISPR-Cas systems and ssODN (single-stranded oligodeoxynucleotide).
CRISPR-Cas systems, in particular, have greatly helped with gene modification in mice and rats. This system involves using Cas9 messenger RNA and gRNA. The gRNA recognizes targeted DNA, and then Cas9 cuts the targeting site. This, effectively, leaves a hole that is then repaired by non-homologous end joining; this causes DNA mutations, resulting in gene knock-out.
Similar to this system is when ssODN is introduced. The same thing occurs, and DNA breaks are repaired through homology-directed repair using donor DNA. However, this results in knock-in of DNA sequences with one to dozens of bases. With ssODN, this allows for the synthesis of oligos of up to 200 bases; however, this makes it difficult to know in large DNA sequences such as green fluorescent protein.
With that said, the new gene modification methods help. With the new methods, the researchers succeeding in achieving efficient and precise knock-in of green fluorescent protein genes. In addition, they were able to replace rat genes with human-derived genes.
Green fluorescent protein is often used to see if a genetic modification "takes." Because it's visible, researchers can see if the organism has incorporated the genetic information that they're aiming for. The ability to use this protein with the new methods makes genetic modification far easier.
So what does this mean for the future? The use of these two knock-in methods will help increase the efficiency of genetic engineering in mice and rats. Since these animals are usually test subjects for genetically-related diseases, this new method could be a huge step forward for designing rats and mice to better test treatments for diseases.
Currently, the researchers hope that their new methods will be able to be used to genetically engineer organisms for drug development, translational research and regenerative medicine.
The findings were published in the Jan. 20 issue of the journal Nature Communications.
