Working on lung development in mouse, you identify a transcription-factor gene that is expressed in developing lungs during embryogenesis. You name this gene LUN1 and decide to investigate its role in lung development. You choose a CRISPR/Cas9 strategy to knock it out. However, you also notice that a second gene named LUN2 also exists in the mouse genome, which encodes a protein that contains a conserved domain with LUN1 at its amino-terminus (Region 1 in the drawing below). The carboxy-terminal end of the protein encoded by this second gene diverges substantially in sequence from the same region (Region 2) in LUN1. The following drawing illustrates the structure of these two genes (please note that the green and blue segments in the legend should be labeled as Region 2, not Region 1 (typo)). Conserved region between LUN1 and LUN2 (Region 1) Gene-specific regions (Region 1) mRNA Promoter t. Terminator A. Considering your first interest is to test the role of LUN1 in lung development, please design a CRISPR/Cas9 construct that will allow you to knock out LUN1: Which region of the LUN1 gene will you choose as a target to synthesize your single guide RNA (sgRNA)? Please explain your answer in a maximum of three short sentences. (6 Points) B. How would you modify this sgRNA gene sequence to allow targeting of Cas9 to both LUN1 and LUN2? Please explain your answer in a maximum of three short sentences. (6 points)..

1. Working on lung development in mouse, you identify a transcription-factor gene that is
expressed in developing lungs during embryogenesis. You name this gene LUN1 and decide to
investigate its role in lung development. You choose a CRISPR/Cas9 strategy to knock it out.
However, you also notice that a second gene named LUN2 also exists in the mouse genome,
which encodes a protein that contains a conserved domain with LUN1 at its amino-terminus
(Region 1 in the drawing below). The carboxy-terminal end of the protein encoded by this
second gene diverges substantially in sequence from the same region (Region 2) in LUN1. The
following drawing illustrates the structure of these two genes (please note that the green and blue
segments in the legend should be labeled as Region 2, not Region 1 (typo)). Conserved region
between LUN1 and LUN2 (Region 1) Gene-specific regions (Region 1) mRNA Promoter t.
Terminator A. Considering your first interest is to test the role of LUN1 in lung development,
please design a CRISPR/Cas9 construct that will allow you to knock out LUN1: Which region of
the LUN1 gene will you choose as a target to synthesize your single guide RNA (sgRNA)?
Please explain your answer in a maximum of three short sentences. (6 Points) B. How would you
modify this sgRNA gene sequence to allow targeting of Cas9 to both LUN1 and LUN2? Please
explain your answer in a maximum of three short sentences. (6 points).