EBK GENETICS: FROM GENES TO GENOMES
6th Edition
ISBN: 9781260041255
Author: HARTWELL
Publisher: MCGRAW HILL BOOK COMPANY
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Chapter 18, Problem 26P
One problem that researchers sometimes encounter when editing genomes with CRISPR/Cas9 is that one or more loci other than the intended target can be recognized by Cas9/sgRNA and cleaved. Part of the reason is that single base pair mismatches between the target site and the sgRNA in the 5′-most half of the 20 bp DNA/RNA hybrid do not prevent Cas9 cleavage of the target site. How could scientists use bioinformatics to avoid such off-target effects?
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Students have asked these similar questions
In order to target a specific region of genomic DNA with CRISPR, researchers must include a guide RNA containing a 20-basepair long spacer sequence that matches the DNA sequence at the target site.
(i) How many possible guide RNA spacer sequences are there?
(ii) One of the possible risks of genetic engineering methods is “off-target” editing, where a modification of the genome occurs in a part of the genome other than the target site. Imagine you design a 20-basepair guide RNA spacer sequence to target a specific portion of the Zebrafish genome, which is 1.7 billion nucleotides long. Assuming all nucleotides are equally common, estimate the probability that your spacer sequence occurs in at least one other position in the Zebrafish genome.
The following illustrates a jagged double-strand DNA break resulting from Cas9 cleavage that
occurred in the first step of genome editing using CRISPR-Cas9 technology:
5'-GCGCCGTCC
3'-CGCGGC
CTGTCAGGCGACACT-3'
AGGGACAGTCCGCTGTGA-5'
Which of the double-stranded DNA sequences listed below (A-D) is expected to result from repair of
the break above via non-homologous end joining? Note: this question is not asking what kinds of
mutations result from NHEJ repair of Cas9 cleavage in general, but specifically what is expected to
result from repair of the jagged cut illustrated above? In the answer choices below, sequences that
are the same in all four options are shown in bold to help you spot the differences.
A. 5'-GCGCCGCTGTCAGGCGACACT-3'
3'-CGCGGCGACAGTCCGCTGTGA-5'
B. 5'-GCGCCGTCTGTCAGGCGACACT-3
3'-CGCGGCAGACAGTCCGCTGTGA-5'
C. 5'-GCGCCGTCCCTGTCAGGCGACACT-3'
3'-CGCGGCAGGGACAGTCCGCTGTGA-5'
D. 5'-GCGCCGAGACTGTCAGGCGACACT-3'
3'-CGCGGCTCTGACAGTCCGCTGTGA-5'
You would like to use a CRISPR-Cas system to knockout a gene that includes the
following sequence:
5'-CATACGAGCGACGACGCATTACGTGGACGTATACACTACATA-3'
3'-GTATGCTCGCTGCTGCGTAATGCACCTGCATATGTGATGTAT-5'
You have designed a guide RNA sequence with the sequence 5'-
UACGAGCGACGACGCAUUACG-3' to work with a Cas9 protein to edit this
sequence. List the sequence of a three nucleotide Protospacer Adjacent Motif
(PAM) that you will need to include in the guide RNA that can be used by this
particular CRISPR-associated protein?
Note: the sequence will be different from the PAM sequence that we were working
with in the CRISPR lab because we are using Cas proteins from other bacteria.
Chapter 18 Solutions
EBK GENETICS: FROM GENES TO GENOMES
Ch. 18 - Match each of the terms in the left column to the...Ch. 18 - Mice are usually gray, but a mouse geneticist has...Ch. 18 - Sometimes, genes transferred into the mouse genome...Ch. 18 - In mice, a group of so-called Hox genes encode...Ch. 18 - The fly eyes shown in Fig. 18.7 are malformed...Ch. 18 - This problem concerns a technique called enhancer...Ch. 18 - Fish and other organisms that live in the Arctic...Ch. 18 - a. Describe two ways you could potentially make a...Ch. 18 - Figure 18.6 shows a picture of Glofish ,...Ch. 18 - Some people are concerned about the possible...
Ch. 18 - The goal of the Knockout Mouse Project is to...Ch. 18 - Prob. 12PCh. 18 - Prob. 13PCh. 18 - a. Which genome manipulation technique would you...Ch. 18 - a. Diagram a knockin construct that could have...Ch. 18 - Prob. 16PCh. 18 - Prob. 17PCh. 18 - The transcription factor Pax6 is required...Ch. 18 - Mouse models for human genetic diseases are...Ch. 18 - One way to determine where inside a cell a protein...Ch. 18 - In Problem 5 in Chapter 17, you saw that a SNP...Ch. 18 - Scientists now routinely use CRISPR/Cas9 to make...Ch. 18 - Geneticists are currently considering using...Ch. 18 - a. Figures 18.9 and 18.12 demonstrated methods to...Ch. 18 - Nonhomologous end-joining NHEJ of a double-strand...Ch. 18 - One problem that researchers sometimes encounter...Ch. 18 - Researchers at the University of California at San...Ch. 18 - Prob. 28PCh. 18 - F. Port and S. Bullock at the University of...Ch. 18 - On Fig 18.14, locate the PAM site and identify the...Ch. 18 - Prob. 31PCh. 18 - Prob. 32PCh. 18 - Recall that Leber congenital amaurosis LCA, a form...Ch. 18 - One potential strategy for gene therapy to correct...Ch. 18 - Recently, scientists have used a mouse model for...
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- What is the difference between nonhomologous end-joining (NHEJ) and homology-directed repair (HDR) in the context of genome editing?arrow_forwardWhy are restriction endonucleases considered a bacteria’s “innate immune system”? Why is CRISPR-Cas9 considered a bacteria’s “adaptive immune system”? What does CRISPR stand for? What is the difference between crRNA and tracrRNA? Why are both needed for Cas9 to function? What does PAM stand for? Where is it found? What is the difference between Non-homologous End Joining (NHEJ) and Homology Directed Repair (HDR)? What is the Guide RNA (gRNA) a chimera of? Why use a gRNA? What new things are researchers doing with CRISPR-Cas9? Reflecting on what you now know about CRISPR-Cas9, what are your thoughts on it’s use in humans and other organisms? What should we be allowed to do? Not do? Are viruses living? Why or why not? What does obligate intracellular parasite mean? What does every virus have? What is the difference between capsomers and…arrow_forwardThe CRISPR-Cas9 system may be able to be used for somatic gene therapy to treat terminal genetic diseases such as Duchenne Muscular Dystrophy (DMD), which affects 1 in 3,500 male births worldwide. As we talked about in class, DMD results from a nonsense mutation that causes a premature stop codon in Exon 23 of the largest gene yet discovered in the human genome, the 79- exon dystrophin gene. Of the choices below, which would be most likely to restore the protein and muscle function? O A deletion of exon 23 O A deletion of intron 23 O A deletion of intron 23 and exon 23 O An insertion of a corrected exon 23 upstream of the mutant exon 23 O A deletion of exons 23, intron 23, and exon 24arrow_forward
- Which of the following is a correct statement about CRISPR-Cas-9 gene editing? Group of answer choices A single guide RNA (sgRNA) recognizes a genomic region followed by 5'-NGG-3' PAM sequence A single guide RNA (sgRNA) recognizes a genomic region followed by a long sequence palindrome repeat A single guide DNA (sgDNA) recognizes a genomic region followed by 5'-NGG-3' PAM sequence PAM sequences induce single stranded breaks that are then repaired by the CAS-9 enzymearrow_forwardYou are studying a gene containing three exons (exon 1 = 66 nucleotides , exon 2 = 99 nucleotides , and exon 3 = 333 nucleotides ) that produces a primary RNA transcript 748 nucleotides long . Gel electrophoresis analysis shows different size mature transcripts are produced in three different cell types . The sizes of the mature transcripts are the following : 1. Neuronal nalcells = 498 nucleotides 2. Muscle cells = 600 nucleotides 3. Liver cells = 399nu nucleotides Primary RNA ranscript = 748 nucleotides a. Name the strategy that allows the same gene to produce different mature transcripts in different tissues . b. Describe the ways (or combinations of exons /introns ) used to produce each one ofthe three mature transcripts . Label the each mature transcripts and include its length . c. Indicate the length of the polypeptide encoded by each mature transcriptarrow_forwardA protein has the following amino acid sequence: Met-Tyr-Asn-Val-Arg-Val-Tyr-Lys-Ala-Lys-Trp-Leu-Ile-His-Thr-Pro You wish to make a set of probes to screen a cDNA library for the sequence that encodes this protein. Your probes should be at least 18 nucleotides in length. Q. How many different probes must be synthesized to be certain that you will find the cDNA sequence that specifies the protein?arrow_forward
- Human genomic libraries used for DNA sequencing are often made from fragments obtained by cleaving human DNA with Haeiii in such a way that the DNA is only partially digested; that is, not all the possible HaeIII sites have been cleaved. What is a possible reason for doing this?arrow_forwardWhat is meant by the term "alignment" in genomic research and in what ways does alignment aid sequence assembly? How can relying on alignment lead to potential errors?arrow_forwardYou isolate a mouse Tau-gene-containing DNA fragment from the chicken and hybridize it to the freshly-made and isolated hnRNA (primary transcript) from the nucleus of the mouse cells transcribed from the Tau gene (immediately after it was produced), allowing no time for processing of the hnRNA. Describe what you see when you look at the DNA/RNA hybrid molecule under the electron microscope.arrow_forward
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