Question

Scientists now routinely use crispr/cas9 to make defined deletions of a gene that can remove several kb of dna from the genome. this method is possible even in cells defective in homologous recombination, as long as the cells can still perform nonhomologous end-joining (nhej).

How could researchers make such deletions?

Answer 1

To make defined deletions of a gene, researchers use CRISPR/Cas9 to create double-strand breaks at specific sites and rely on nonhomologous end-joining (NHEJ) to rejoin the ends, which results in the deletion of the desired DNA sequence.

Step-by-step explanation:

Researchers can utilize the CRISPR/Cas9 genome editing system to create specific deletions in DNA, even in cells that are defective in homologous recombination. The technique involves designing a guide RNA (gRNA) that targets the specific site in the genome where the deletion is desired.

Following the introduction of the CRISPR/Cas9 complex into the cell, it creates a double-strand break (DSB) at this location. In cells capable of nonhomologous end-joining (NHEJ), this repair pathway can be exploited to rejoin the DNA ends after a deletion occurs. By using two gRNAs that flank the region to be deleted, the intervening sequence can be excised when Cas9 induces DSBs at both sites. The NHEJ process, which is error-prone, can then rejoin the ends, resulting in a deletion of several kilobases (kb) of DNA without the need for homologous recombination.