Question

the following scenarios require application of CRISPR-cas9 technology. Identify which technologies are the best fit for each research scenario

Cas9 and HDR 1. Researchers want to make an endonuclease that binds to target DNA, but does not cut the DNA. This will allow them to target specific areas of the genome for other modifications without making cuts. dCas9 dCas9 and repression domain 2. Researchers are studying a proposed oncogene in mice. They want to examine what happens when the gene is overexpressed spressed dCas9 and activation domain dCas9 and fluorescent protein 3. Researchers are studying a proposed tumor suppressor gene in fruit flies. They want to examine what happens when the gene has lower than normal expression, without inactivating the gene entirely. Cas9 and NHEJ 4. Researchers are studying the three-dimensional organization of genomic structure. They want to visualize the location of specific genomic loci within a living cell. 5. Researchers identify some previously uncharacterized genes that might play a role in diabetes. They want to make mouse mutants with null mutations in those genes and study what symptoms arise as a result 6. Researchers are studying an allele associated with a longer life span in humans, which varies from the normal variant by six base pairs. In order to study this allele in the laboratory, they want to modify the mouse homologue to have the same 6 base pair variation.

Answer 1

1. Researchers want to make an endonuclease that binds to the target DNA but does not cut the DNA. This will allow them target specific areas of genome for other modifications without making cuts. dCas9 - Also called dead Cas9, a mutant Cas9 whose endonuclease activity has been removed with point mutations in its endonuclease domain.

2. Researchers are studying a proposed oncogene in mice. They want to examine what happens when the gene is over expressed. dCas9 and activation domain- This activation domain helps in the activation of transcription by recruiting general transcription factors or RNA polymerase to the site of gene of interest.

3. Researchers are studying a proposed tumor suppressor gene in fruit flies. They want to know what happens when the gene has lower than normal expression, without inactivating the gene entirely. dCas9 and repression domain- This is also called CRISPR interference which repress transcription at transcription initiation or elongation step by blocking it.

4. Researchers are studying three dimensional organization of genomic structure. They want to visualize the location of specific genomic loci within a living cell. dCas9 and Fluorescent protein. This Fluorescent protein will help in tagging speific loci within a living cell.

5. Researchers identify some previously uncharacterized gene that might play a role in diabetes. They want to make mouse mutants with null mutations in those genes and study what symptoms arise as a result. Cas9 and NHEJ

6. Researchers are studying an allele associated with a longer life span in humans, which varies from the normal variant by six base pairs. In order to study this allele in the laboratory, they want to modify the mouse homologue to have the same 6 base pair variation. Cas9 and HDR- Through this the six base pairs can be inserted into mouse due to homologous sequence in human and mouse.