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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.

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.

Human Heredity: Principles and Issues (MindTap Course List)
Human Heredity: Principles and Issues (MindTap Course List)
11th Edition
ISBN:9781305251052
Author:Michael Cummings
Publisher:Michael Cummings
Chapter13: An Introduction To Genetic Technology
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Problem 20QP: Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant...
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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.
Transcribed Image Text: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.
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