Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

16 - Genome Editing

Genome editing is a technique that introduces targeted changes to the DNA of an isolated cell or an entire organism.  Genome editing allows scientists to insert a DNA sequence, delete a DNA sequence, or modify the DNA sequence of any gene within the genome. The goal of genome editing is to change the phenotype of a cell in a way controlled by the researcher.

The applications of genome editing are staggering.  Genome editing can be used in research to better understand the role of a gene and its protein product in cellular structure and function.  Genome editing can also be used as a treatment for genetic diseases by replacing a mutant allele that causes the disease with a wild-type allele. Genome editing can also enhance the yield of crops or give desirable traits to livestock.

Key Questions

  • What is meant by genome editing?
  • What are some of the applications of genome editing? 

Genome editing (overview)

Genome editing works by recognizing a specific DNA sequence (i.e., the target DNA sequence) in the genome.  After recognition of the target DNA sequence, an endonuclease cuts the backbones of both DNA strands. The cell tries to fix the dsDNA break by rejoining the two ends of the severed DNA molecule; however, the repair mechanism involved is error-prone, introducing extra nucleotides or deleting nucleotides at the cut site.  The insertion or deletion of a single base or two bases within the coding region of a gene changes every codon downstream of this insertion/deletion (indel) site.  This type of mutation, referred to as a frameshift mutation, produces a defective protein product (see Part 7).

Key Questions

  • How does genome editing work?
  • What is meant by an indel and a frameshift mutation?

Genome editing systems

There are three major genetic technologies that can be used to edit DNA sequences within isolated cells or entire organisms:

ZFNs and TALENs have many drawbacks, including the high cost and time involved in engineering the DNA binding domains within the nucleases, inefficient cutting of the DNA, and the difficulty of working with these systems. Although the ZFNs and TALENs have been used to successfully edit genes, the science world has embraced CRISPR-Cas9 due to its lower cost, higher efficiency, and potential to create multiple genome edits simultaneously.  Because of its widespread current use and promising future, CRISPR-Cas9 will serve as the subject for the remainder of this chapter.

Key Questions

  • Describe the three major genome editing technologies.
  • Why do scientists prefer CRISPR-Cas9? 

The CRISPR-Cas9 genome editing system

CRISPR is an acronym for the clustered regularly interspaced short palindromic repeats (CRISPR) system.  The CRISPR-Cas9 system has two molecular components (see figure 16.1):

The DNA sequence targeted by the sgRNA needs to contain a protospacer adjacent motif (PAM) sequence, as Cas9 binds to the PAM sequence to position itself while it cuts both strands of the DNA.  The PAM sequence is a DNA consensus sequence consisting of 5'-NGG-3', where N is any one of the four DNA bases (A, T, C, or G). The PAM sequence is in the nontarget DNA strand; the nontarget DNA strand does not form hydrogen bonds with the crRNA component within the sgRNA.  The PAM in the nontarget DNA strand is located 3-4 nucleotides in the 3’ direction from the site that will be cut by Cas9. 

The CRISPR-Cas9 system creates genome edits as follows:

  1. Cas9 binds to the PAM sequence in the nontarget DNA strand.
  2. The target and nontarget DNA strands are separated from each other. The Cas9 enzyme is the helicase that separates the two DNA strands.
  3. The crRNA attempts to form hydrogen bonds with the target DNA strand. If the crRNA forms proper hydrogen bonds with the target DNA strand, then genome editing continues.  If hydrogen bonds fail to form, the Cas9 enzyme is released and binds to another PAM sequence in the genome.
  4. The binding of the crRNA to the target DNA strand activates tracrRNA, which in turn, activates Cas9. 
  5. Cas9 enzyme cuts both DNA strands 3-4 nucleotides in the 5’ direction (along the nontarget strand) from the PAM site.
  6. Once both strands of the DNA have been cut by Cas9, the cell's DNA repair systems attempt to fix the error and, in doing so, can add a few bases, delete a few bases, or insert a completely new piece of DNA.
Figure 16.1 The CRISPR-Cas9 System --- Image created by SL

Key Questions

  • What is meant by the target and nontarget DNA strands?
  • What are the names of the two components within a sgRNA molecule?
  • Describe how crRNA, tracrRNA, Cas9, and the PAM contribute to the CRISPR-Cas9 genome editing system.

What is the natural function of CRISPR-Cas9?

The CRISPR-Cas9 system is thought to be analogous to a bacterial immune system, protecting bacteria against invading bacteriophages (viruses that infect bacteria). The CRISPR gene locus in bacteria consists of clusters of repetitive DNA sequences (short palindromic repeats that are 30-40 base pairs in length) separated by bacteriophage DNA sequences called spacers.

Insert Figure 16.2 - CRISPR protects bacteria from reinfection with bacteriophages.

During an infection, the bacteriophage genome is injected into the cytoplasm of the bacterial cell.  If the bacterium survives the encounter with the bacteriophage, the bacteriophage DNA is cut by nucleases, and a portion of the bacteriophage genome is stored in the CRISPR gene locus as a spacer DNA sequence. In essence, the CRISPR locus in a bacterium is a library of previous bacteriophage infections. 

Upon reinfection with the same bacteriophage, the CRISPR gene locus is transcribed to produce two types of RNA molecules.  The spacer DNA sequence is transcribed to produce the single-stranded CRISPR RNA (crRNA) that forms hydrogen bonds with the DNA of the infecting bacteriophage.  Another gene in the CRISPR locus is transcribed to make the transactivating crRNA (tracrRNA). The crRNA and the tracrRNA from hydrogen bonds with each other and then bind to the Cas9 endonuclease.  Note that the tracrRNA contains the stem-loop that activates Cas9.  The tracrRNA:crRNA:Cas9 complex then binds to a PAM sequence in the DNA of the invading bacteriophage.  The two DNA strands within the bacteriophage DNA are separated and the crRNA forms hydrogen bonds with the target DNA strand, while the nontarget DNA strand is moved out of the way.  Finally, the Cas9 protein makes double-stranded breaks (DSB) in the DNA of the bacteriophage, thereby destroying the bacteriophage genome and inhibiting the phage infection.

Key Question

  • How is the CRISPR-Cas9 system beneficial to a bacterial cell?
  • How are spacers generated?
  • Describe how the CRISPR-Cas9 system destroys the DNA of an invading bacteriophage.

Applications of CRISPR-Cas9

Genome editing via CRISPR-Cas9 is as easy as designing a crRNA that consists of a 20 nucleotide-long RNA sequence that forms hydrogen bonds with any target DNA sequence of interest. This crRNA is covalently linked to the tracrRNA that forms an RNA stem-loop.  The crRNA linked to the tracrRNA is the sgRNA.  Both the sgRNA and Cas9 DNA sequences are ligated into separate cloning sites within a vector, and the vector is introduced into a eukaryotic cell.  Activation of the vector in eukaryotic cells will result in the production of both the sgRNA and Cas9 molecules.

When the sgRNA binds to a target DNA sequence, Cas9 will produce a double-stranded break (DSB) in the DNA. When the cell attempts to repair these DSBs, the cell can undergo the non-homologous end joining (NHEJ) DNA repair pathway. NHEJ is not perfect, and insertion or deletion of a few nucleotides occurs (these mutations are called indels).  Recall that indels cause a frameshift in translation that ultimately prevents the eukaryotic gene from making a functional protein product.  Therefore, CRISPR-Cas9 genome editing followed by NHEJ allows the researcher to produce a gene knock-out that fails to produce the encoded protein.

Double strand breaks in the DNA can also lead to another type of DNA repair known as homology directed repair (HDR). In this case, DNA repair allows the insertion of a donor sequence at the location of the DSB, instead of repairing the break by inserting or deleting a few nucleotides. The donor DNA can be engineered to contain a known gene mutation. This approach allows the researcher to insert a mutant gene in the place of a wild-type gene to study the effects of the mutation on the cell.  Alternatively, the donor DNA sequence can contain a wild-type version of a gene that can replace the mutant form of the gene within the cell.  The replacement of a gene with a different allele of the same gene produces a knock-in cell.

CRISPR-Cas9 is a convenient genome editing system to use because if you wish to study a different gene, all you do is design a new 20 nucleotide crRNA that forms hydrogen bonds with the new target gene, all of the other components (tracrRNA and Cas9) of the system remain the same.  Moreover, the use of multiple unique crRNA sequences allows the alteration of several genes in the genome simultaneously.

Key Questions

  • How is gene cloning used in genome editing?
  • Describe how NHEJ can be used to create a knock-out cell.
  • Describe how HDR can be used to create a knock-in cell.

Challenges associated with CRISPR-Cas9

Before we explore the ethics of using the CRISPR-Cas9 system for genome editing, let us investigate some of the challenges of using the CRISPR-Cas9 system.  In a typical experiment, the researcher will introduce the CRISPR-Cas9 vector into a population of eukaryotic cells. Because the process of genome editing is inefficient, the experiment will result in three groups of cells in the population: those in which no editing occurred, those which have one of the two copies of a gene edited, and those with both alleles edited.  If the knock-out approach is used, the researcher will want to study cells that have no functional copies of the gene; therefore, the researcher will select those cells with both alleles edited. DNA sequencing is one of the easiest ways to confirm that the desired changes have taken place.

The sgRNA is designed for a specific sequence in the genome; however, sometimes a 20 nucleotide-long crRNA can bind to more than one DNA sequence in the genome of the target cell. This raises the possibility that the CRISPR-Cas9 system will cut the DNA at undesired locations within the genome, producing off-target effects.  Because the locations of these off-target cut sites are difficult to predict, treating cells with CRISPR-Cas9 can have unintended consequences on the cell.

Key Questions

  • What are two challenges associated with CRISPR-Cas9 genome editing?

The ethics of genome editing in humans

Many scientists are interested in using CRISPR-Cas9 to treat human genetic diseases, especially diseases for which there is currently no treatment. There are two main ways that human genome editing can be used to treat disease: inactivation of a mutant gene to remove its effects on the cell (using the NHEJ knock-out approach) or insertion of a functional allele to replace a mutant one (using the HDR knock-in approach). 

With the promise that CRISPR-Cas9 brings, there is also uncertainty about the ethics of this technique, particularly when applied to humans. Most researchers agree that if we have the tools necessary to treat a genetic disease, then we should use those tools to improve the lives of patients. However, considerable disagreement exists as to whether the CRISPR-Cas9 technique should be used to modify gametes-producing cells or embryonic cells.  Current laws in the United States prohibit the use of human genome editing in gamete-producing cells. Research on human embryos is permitted if the treated human embryos are destroyed before day 14 of development and are not implanted into the womb.

An important issue to consider with genome editing is that of informed consent. An adult can give consent for genome editing that can potentially treat a genetic disease, but when that treatment extends to embryonic genome editing, there is no way to obtain consent from the embryo. Public opinion remains divided as to who has the right to make the decision; is it the person who will be affected by the change or the society who is caring for that individual?

In November 2018, the press announced that a researcher in China used CRISPR-Cas9 to successfully edit the CCR5 gene in twins (one received the edit while the other did not). Knocking out this gene is expected to prevent the treated child from contracting an human immunodeficiency virus (HIV) infection, even when exposed to HIV (their biological father was HIV positive). To say the scientific world was upset about this announcement would be an understatement. This was the first time that a human baby was born after genome editing was performed. The reason why this announcement was not received with congratulations was, in part, due to the lack of informed consent and the failure to make sure that no off-target effects took place before implanting the embryos into the birth mother. In fact, it is uncertain if the parents were informed as to the genome editing experiment, or if they were coerced into giving their consent.

There may never be an international agreement concerning genome editing that can be enforced by all nations. Even when there is agreement as to what is ethical and what is not, there will always individuals or poorly regulated nations who will carry out research that is contrary to the moral beliefs of others around the world. Important questions to consider include how do we establish laws concerning the ethical practice of scientific research, and how do we penalize those who knowingly disobey those laws?

In 1956, mathematician and biologist Jacob Bronowski wrote that, as scientists, “We ought to act in such a way that what is true can be verified to be so,” an expression of his belief that it is our right and our duty to explore the unknown. The point we are trying to make here is that maybe having large international committees decide what should be practiced and what should be prohibited is not the real question, but rather how can we ensure that research is based on sound scientific principles? Decide what research is worth pursuing, then carry out that research as you believe appropriate. The scientific knowledge of the world is only as strong as the ethical behaviors of those practicing it.

Key Questions

  • Should genome editing be done on gamete-producing cells, embryonic cells, or somatic cells? Why or why not?

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