CRISPR: A revolution in Genome Editing

The following scenarios require application of CRISPR-Cas9 technology. Identify which technologies are the best fit for each research scenario.

1. dCas9 2. dCas9 and activation domain 3. dCas9 and repression domain 4. dCas9 and fluorescent protein 5. Cas9 and NHEJ 5. Cas9 and HDR

What steps could help refine the researchers' experiment to modify only the two target genes? Select all that apply. 1. Engineer a version of Cas9 that demonstrates higher fidelity to the target sequence. 2. Inject the plasmids into younger mouse embryos so more cells are modified. 3. Insert more Cas9 and sgRNA during the initial introduction of the components. 4. Make stable transgenic lines so the cutting by Cas9 is not transient. 5. Screen the rest of the mouse genome to be sure the 20-bp targets do not appear elsewhere in the genome.

Answer: Engineer a version of Cas9 that demonstrates higher fidelity to the target sequence. Screen the rest of the mouse genome to be sure the 20-be targets do not appear elsewhere in the genome.

Complete the following sorting exercise to be sure you understand how the two break repair mechanisms are similar and different.

NHEJ Repair ONLY 1. More error-prone; likely to cause indels and frame shift mutations. 2. Often results in nonfunctional (null) mutants. 3. Simply involves ligation of broken DNA fragments. HDR repair ONLY 1. Better for making complex substitutions. 2. Increases in frequency after single-strand breaks. 3. Ses an underaged homologous chromosome as a template. Both NHEJ and HDR Repair 1. Occurs after a DNA break (including breaks made by Cas9).

The following figure shows the general steps that occur when a researcher uses the CRISPR-Cas9 system to modify a protein-encoding gene in a eukaryotic cell with the goal of modifying the protein product. Drag the descriptions of the steps to their appropriate locations on the figure.

1. Create plasmid expression vectors with genes encoding Cas9 and sgRNA. 2. Introduce the vectors into a eukaryotic cell. 3. The gene expression machinery of the cell produces the Cas9 protein and the sgRNA. 4. The Cas9 protein, guided by sgRNA, binds to its target sequence in the cell's DNA. 5. The Cas9 protein cleaves the target sequence. 6. During the process of DNA break repair, the target sequence may be modified. 7. The modified DNA sequence is transcribed into RNA. 8. The RNA is translated into a modified protein.