CRISPR/Cas9-guide RNA complexes hold enormous promise as aids for genome engineering in plants and animals. The CRISPR system is almost too good to be true. You want to test just how specific the Cas9-guide RNA complexes are; that is, whether they really recognize individual sites in the genome, which is the basis for their touted actions. You realize that you can test their specificity using the "DNA curtain" assay you have developed. You make the DNA curtain by tethering single molecules of bacteriophage lambda DNA (about 50,000 nucleotides) at one end, and then stretching them in the same direction by flowing buffer across the slide. You incubate the DNA curtain with a highly fluorescent version of Cas9-either loaded with the guide RNA or free-and visualize the distribution of the Cas9 by sensitive fluorescence microscopy, as shown the figure below. A. As described in Jinek et al., what are the essential nucleic acid features that specify where Cas9 will cut? (please use six or less sentences in your answer) B. Phage lambda DNA has a single site that perfectly matches the guide RNA. Does your experiment support the existence of a single site that is recognized by the Cas9-guide RNA complex? Explain your answer. C. One of the main concerns for using the CRISPR system is that the Cas9- guide RNA will bind to other sites that are related to the intended target (so-called off-target effects). Is there any evidence for off-target binding in your experiment? Do you think the results would be any different if you had a DNA curtain made from human chromosome 1 (about 250,000,000 nucleotides)? (A) RNA-loaded Cas9 12 5 µm | (B) RNA-free Cas9 5 μη! Figure Legend: DNA curtain assay for target binding by Cas9-guide RNA. (A) DNA curtain incubated with Cas9-guide RNA that matches one site in the lambda genome. Pink spots indicate the sites where Cas9-guide RNA is located. (B) DNA curtains incubated with Cas9 in the absence of guide RNA.

The prospect of using gene therapy to alleviate genetic conditions is still a vision of the future. Gene therapy for adenosine deaminase deficiency has proved to be quite promising, but many obstacles remain to be overcome. Currently, the correction of human genetic defects is done using retroviruses as vectors. For this purpose, viral genes are removed from the retroviral genome, creating a vector capable of transferring human structural genes into sites on human chromosomes within target-tissue cells. Do you see any potential problems with inserting pieces of a retroviral genome into humans? If so, are there ways to combat or prevent these problems?

In selecting target cells to receive a transferred gene in gene therapy, what factors do you think would have to be taken into account?

Figure 17.7 You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine. What results would you expect from your molecular cloning experiment? There will be no colonies on the bacterial plate. There will be blue colonies only. There will be blue and white colonies. The will be white colonies only.