02 - Code Breakers: Using CRISPR to Rewrite Genetics
NHEJ s a simple but error-prone process that occurs naturally in cells and is used in the lab for knock- out experiments. These are experiments that study the function of a gene by inactivating it with a loss of function mutation and then observing the results. When Cas cuts through the DNA backbone, the cell’s most direct means of repair is to join the two broken ends of DNA back together. However, this process usually results in small deletions or insertions at the cut site which cause frameshift mutations that disrupt the function of the targeted region. Unlike classical knockout methods, which required screening thousands of random mutations, CRISPR knockout experiments allow research- ers to design guide RNA that automatically targets their gene of interest, streamlining the process by eliminating one of the most laborious and time-intensive steps. HDR is also a naturally occurring process but more precise, controlled, and complex than NHEJ. It involves mending the double-stranded break using a donor or template DNA as a reference. To work properly, this template DNA must contain regions that match the target DNA sequences on both sides of the break. In cells, this is often the sister chromosome, which shares the same sequence. In CRISPR experiments, researchers develop a repair template that matches the genomic DNA around the cut site. However, in between these homologous beginning and ending regions, scientists can introduce specific mutations or new genes. As the cell repairs itself, these insertions are transcribed from the template to the target DNA allowing for specific and user-designed changes to an organ - ism’s genome.
Figure 4 - Repairing DNA Using HDR
CRISPR-Cas targeting DNA
Corrected DNA sequence
Homologous DNA sequences
Homology Directed Repair (HDR)
Repaired DNA
Figure 4: Repairing DNA Using HDR
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