1. Introduction: The Brave New World of Human Genetic Modification is Here

On November 25, 2018, Dr. He Jiankui, a scientist working in China, announced that he had created the first genetically edited human babies. Dr. He’s claim (still unverified) is that he used a technology known as CRISPR-Cas9 to disable a gene called CCR5 in two twin girls, increasing their resistance to infection by HIV.

Figure 0: H.I.V. uses the CCR5 protein to infect Helper T cells

The CCR5 gene codes for a receptor protein (also called CCR5) that HIV (the virus that causes AIDS) uses to infect cells. In Phase 1 at left you can see HIV bumping into a Helper T Cell. This cell is like the general in our immune system’s army. HIV has a surface protein, gp120, shown at “b,” that binds with a protein called CD4 (letter “e”) on the membrane (c) of the Helper T cell. In Phase 2, the bound CD4 protein forms a complex with the CCR5 protein (at “d”). The formation of this complex induces receptor mediated endocytosis (shown at Phase 3). In subsequent phases (not shown) HIV integrates itself into the Helper T cell’s nucleus, transforming the cell into an HIV factory, and ultimately killing the T-cell (and destroying a person’s immune system in the process). Click here for my tutorials about the immune system, and here for a tutorial about the life cycle of H.I.V. All links on this page will open in a separate tab.)

Many humans (including about 10% of Europe’s population) have a natural resistance to HIV infection because they carry a mutation in the CCR5 gene. That mutation results in a much smaller version of the CCR5 protein that no longer sits on the outside of the membrane, blocking entry of HIV into helper T cells (see HIV Resistant Mutation at Nature SciTable)

The twins were born to a Chinese couple in which the father was HIV positive. Dr. He explains in this youtube video that his motive was to protect the girls from HIV infection and subsequent discrimination, and that the birth of these girls had given the twins’ father “a reason to live.”

The response to Dr. He’s work has been worldwide condemnation (see this report from the Associated Press, and this article in the NY Times). Why? Human gene editing of any type opens up a host of ethical issues, and Dr. He had taken it upon himself to cross a line that humanity hasn’t come to any clear agreement about. Additionally, the type of edit that Dr. He carried out was the one that people worry most about. It wasn’t merely a change in the cells of one body tissue to cure a disease (a proposed therapy that I’ll describe below). Rather, Dr. He had changed the genome of every cell in the body of these twins, including their germ cells. If these girls reproduce, they can pass on the edited gene to their offspring. That kind of change brings up questions for which there are no clear answers:

  • What are the conditions (if any) that justify human genetic modification? For example, is it okay to do gene-editing to prevent a couple from passing on a genetic disease like hemophilia or cystic fibrosis to their children? Is it okay for a couple to ensure that their child will have curly hair? Brown eyes?
  • What are the risks that genetic editing brings to the offspring?
  • What will the socio-economic consequences of genetic editing be? Are we paving a path to a world where there will be those who can afford to genetically modify their children, and those who can’t? Are there ways to ensure that the benefits of this technology are used wisely, and distributed in a way that makes our world more just and equitable?

So those are the big issues. But even on a much more  superficial and technical level, Dr. He’s work was medically unjustifiable. First, the girls were not at risk for HIV infection, and editing their genomes to prevent expression of the CCR5 gene was unnecessary. Many well-established measures could have been used to protect them from HIV infection in utero and during birth. Second, genetic modification entails unknown risks: it’s possible that other genes in the girls’ genomes might have been affected (though Dr. He has made assurances that this is not the case). Third, in one of the twins the gene edit was only partially successful: one allele still contains the working CCR5 gene, meaning that she’ll express the protein, and be just as much at risk of contracting HIV as an adult as anyone else. 

With CRISPR-Cas9, modifying the genome of any organism — humans included — has become easier than ever before. To get an overview of how CRISPR works, watch the video below, and then answer the questions that follow.

2. Overview Video of CRISPR-Cas9

 

3. CRISPR-Cas9 Video Quiz

[qwiz qrecord_id=”sciencemusicvideosMeister1961-CRISPR 1 (Video) M17″]

[h]Genome editing with CRISPR-Cas9: Video Quiz

[i]

[q] CRISPR first evolved as a system that bacteria used to protect themselves from attack from [hangman].  

[c] viruses

[q] When CRISPR containing bacteria are attacked by a virus, they produce two types of[hangman] . These molecules form a complex with a protein that’s called [hangman].

[c] RNA

[c] cas9

[q multiple_choice=”true”] Cas9 is a nuclease. That means that it’s an enzyme that’s capable of cutting up

[c] phospholipids

[f] No. Think about what viruses inject into cells when they attack them.

[c] proteins

[f] No. Think about what viruses inject into cells when they attack them.

[c*] nucleic acids

[f]Excellent! Cas9 is a nuclease, an enzyme capable of cutting up DNA.

[q] CRISPR-Cas9 uses guide [hangman] to find matching DNA from a [hangman]. When it does, it makes a double-stranded cut in the viral [hangman], which disables the virus and keeps it from destroying the cell

[c] RNA

[c] virus

[c]DNA

[q]In recent years, scientists have engineered the CRISPR-Cas9 system so that instead of only cutting DNA from a [hangman], it can cut any targeted sequence of [hangman] in any organism. All that was required was changing the [hangman] RNA.

[c]virus

[c]DNA

[c]guide

[q multiple_choice=”true”] After the Cas9 enzyme cuts up targeted DNA in a cell, the cell

[c] initiates cell suicide pathways (apoptosis) that result in lysosomal rupture and eventual cell death.

[f] No. Apoptosis is a real phenomenon, but cells usually save that move as a last resort. Next time, choose another answer.

[c*] reconnects the broken DNA, but often in a way that results in mutations that disable the gene.

[f] That’s exactly right. Cells respond to DNA breaks through DNA repair mechanisms.

[c] starts dividing out of control, often becoming a tumor that could develop into cancer.

[f]No. There is a connection between mutation and cancer, but there’s a better answer.

[q]If scientists add working DNA to a cell where CRISPR-Cas9 is at work, the cell might cut out a defective gene and [hangman] in a working copy of that gene (hint: think of how you cut and ______ words in a word processor).

[c]paste

[q]If CRISPR-Cas9 and foreign [hangman] is introduced to a fertilized egg, it can be used to produce transgenic organisms.

[c]DNA

[/qwiz]

4. Understanding CRISPR

As you just saw, CRISPR-Cas9 evolved as a molecular defense used by bacteria to fight off attack from bacteriophage (viruses that attack bacteria).

Figure 1: The Lytic Cycle

Bacteria are constantly attacked by viruses (a process that you can review through this tutorial). For now, all you need to know is what’s called the lytic cycle (shown at left). When a virus (“a” and “b”) attacks a bacterial cell (d), it injects its DNA (e) into its bacterial victim. The viral DNA converts the bacterial cell into a virus factory (steps 3 and 4)  that uses the cell’s molecular machinery (polymerases and ribosomes) to assemble new viruses (step 5), which eventually burst out the cell (step 6).

Bacteria have evolved countermeasures to fight back. These include restriction enzymes, and also the CRISPR-Cas9 system. Both systems are capable of latching onto injected viral DNA and destroying it by breaking it apart. The CRISPR-Cas9 system, however, not only beats back invaders by destroying viral DNA: it’s also a part of system that remembers viral invaders, and thereby acts as a bacterial version of the vertebrate acquired immune system.

CRISPR is an acronym that stands for clustered regularly interspersed short palindromic repeats. Along a bacterial chromosome (“c,” above, “A” below) are clusters of repeated sequences (B), interspersed with what are called “spacers” (C). The spacers are non-repetitive DNA, and they consist of fragments of invading plasmid or viral DNA.

Figure 2: The CRISPR Array. Adapted from an image presented by Jennifer Doudna, credited to Jillian Banfield, UC Berkeley. Also credited to Bolotin, Mojica, and Pourcel, 2005.

How did this viral DNA become integrated into a bacterial chromosome?

Figure 3: CRISPR Immunization: Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051438/. Permission Pending. Click the image to view a larger version in a new tab.

 

 

The image at left shows a phage (a) injecting its DNA (c) into a bacterial cell (b). Nuclease enzymes that are part of a CRISPR associated complex (also known at Cas Complex enzymes, shown at “d”) attack the viral DNA, breaking it into fragments (e). One of these fragments (f) becomes incorporated into the CRISPR array (j) as a new spacer (g). Letter “i” shows spacers that were incorporated into the CRISPR array after previous infections. “H” shows repeats.

The Cas nuclease complex shown above at “d” is a kind of innate immunity. It’s part of a bacterium’s generalized arsenal for fighting off bacteriophages. Once the new spacer (g) has been integrated into the CRISPR array, however, the cell has a kind of acquired immunity against further attack from this virus. Here’s how this works. Note that you have to scroll the text below the diagram to read the entire explanation.

Figure 4: CRISPR-Cas9 Immunity: Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051438/. Permission Pending. Click the image to view a larger version in a new tab.
  • STEP 1: A phage (at “a”) attacks the cell by injecting its DNA. Note that an identical phage had previously attacked this cell (or one of its ancestors), and a spacer with a fragment of this phage’s DNA is in the cell’s CRISPR DNA (at bottom right).
  • STEP 2: Because a clone of this phage had previously attached the cell, the cell now has a new weapon with which to counterattack. The cell transcribes its CRISPR DNA into pre-CRISPR RNA. Note that CRISPR is an operon: a series of genes that gets transcribed together into a single RNA transcript, labeled in this diagram as “pre-CRISPR RNA.”
  • STEP 3: The cell’s counterattack requires transcription of additional genes. One of these is the  Cas9 gene (shown a little bit to the right and below number 3). As we learned above, Cas9 is a nuclease. With proper guidance from CRISPR RNA, it will be able to attack and destroy the viral DNA that’s been injected into the cell. An additional gene is the tracrRNA gene, which is transcribed into tracrRNA. As you can see below, tracrRNA bonds with mature CRISPR RNA to form a functional CRISPR-Cas9 complex. In some sources, it’s described as a “handle” that Cas9 holds on to as it carries out viral target surveillance and destruction. Study the image below to see all of the components of CRISPR-Cas9.
  • STEP 4: Multiple CRISPR-Cas9 complexes form as the Cas9 protein, the RNA transcribed from the spacer DNA, and the tracrRNA all come together. Once associated with Cas9, the spacer RNA now graduates to become crRNA. The crRNA is the guide that the complex uses to find injected phage DNA. RNAase III is yet another enzyme that edits the various RNAs into their mature forms.
  • STEP 5: Of the various CRISPR-Cas9 complexes built, one has the crRNA that complements a 20 nucleotide sequence in the DNA from the invading phage. That stretch of recognizable DNA is called a protospacer.
  • STEP 6 : When the CRISPR-Cas9 encounters phage DNA, the viral DNA is split open and lined up against the crRNA. If the crRNA complements the protospacer in the viral DNA…
  • STEP 7: …then the viral DNA is broken apart, preventing the virus from taking over the cell.

5. Understanding CRISPR-Cas9: Interactive Diagrams

[qwiz style=”width: 700px !important;” qrecord_id=”sciencemusicvideosMeister1961-CRISPR 2, Interactive Diagrams (M17)”]

[q labels = “top”]

 

[l]Cas Nucleases

[fx] No. Please try again.

[f*] Excellent!

[l]cell wall/membrane

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]Fragmented Viral DNA

[fx] No. Please try again.

[f*] Correct!

[l]Insertion of New Spacer

[fx] No, that’s not correct. Please try again.

[f*] Correct!

[l]Newest spacer

[fx] No. Please try again.

[f*] Correct!

[l]Older spacers

[fx] No. Please try again.

[f*] Correct!

[l]phage

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]Repeats

[fx] No. Please try again.

[f*] Great!

[l]viral DNA

[fx] No. Please try again.

[f*] Excellent!

[!]Second Diagram [/!!!]

[q labels = “top”]

 

[l]Cas9 

[fx] No. Please try again.

[f*] Great!

[l]Cas9 genes

[fx] No. Please try again.

[f*] Good!

[l]Cell membrane/wall

[fx] No. Please try again.

[f*] Excellent!

[l]CRISPR DNA

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]crRNA

[fx] No. Please try again.

[f*] Great!

[l]Fragmented Viral DNA

[fx] No. Please try again.

[f*] Good!

[l]Mature CRISPR-Cas9

[fx] No. Please try again.

[f*] Excellent!

[l]pre-CRISPR RNA

[fx] No, that’s not correct. Please try again.

[f*] Correct!

[l]Phage

[fx] No. Please try again.

[f*] Correct!

[l]repeats

[fx] No. Please try again.

[f*] Great!

[l]spacers

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]tracrRNA

[fx] No. Please try again.

[f*] Correct!

[l]tracrRNA genes

[fx] No. Please try again.

[f*] Correct!

[l]Viral DNA

[fx] No. Please try again.

[f*] Good!

[!]Third Diagram [/!!!]

[q labels = “top”]

 

[l]Cas9

[fx] No. Please try again.

[f*] Good!

[l]crRNA

[fx] No. Please try again.

[f*] Great!

[l]tracrRNA

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]viral DNA

[fx] No, that’s not correct. Please try again.

[f*] Correct!

[/qwiz]

6. CRISPR-Cas9 Becomes a Genome Editing System

By 2012, the  mechanisms involved in the CRISPR-Cas9 system were well understood. This included understanding how CRISPR-Cas9 can target viral DNA (the protospacer), but not target the spacer DNA derived from protospacer/viral DNA. The key to this involves a short stretch of DNA known as the PAM, for Protospacer Adjacent Motif. The PAM is a 2-6 base pair DNA sequence, such as 5′-NGG-3′ (two guanine nucleotides, preceded by any nucleotide base (N) at the 5′ end). In CRISPR-Cas9 systems found in Staphylococcus pyogenes (a bacterial species in which CRISPR systems were widely studied), Cas9 will only cut viral DNA that contains this sequence. The genomic trick that’s carried out by bacterial systems with CRISPR-based immunity is that the spacer sequence does not contain the PAM, protecting the bacterial cell’s own DNA from its Cas9 enzymes. (various sources, included Wikipedia and this youtube video.)

So if we look again at CRISPR-Cas9, we can now understand all of its parts.

Figure 5: Wild-type CRISPR-Cas9

The Cas9 protein is indicated by letter “g.” Letter “e” is the tracrRNA (the handle), and “f” is crRNA, which includes the spacer RNA  that recognizes the protospacer sequence (c) in the invading viral DNA (a).  Letter “d” is the PAM sequence, the molecular signal that tells Cas9 that this is viral RNA and should be cut. Letter “b” (which appears twice) represents the nucleases that make a double stranded cut in the viral DNA.

With the science understood, a flash of insight followed. If CRISPR RNA enables the CRISPR-Cas9 system to recognize viral DNA and make a double stranded cut, then why not re-engineer crRNA to target any DNA sequence? This insight emerged in several labs and is credited to various scientists, including Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, and George Church.

Figure 6: Engineered CRISPR-Cas9 with single guide RNA (sgRNA)

The CRISPR-Cas9 used in genetic engineering has been simplified from its form in nature by combining the tracrRNA with the crRNA into a single strand that’s called single guide RNA (sgRNA). This sgRNA is shown at “f”. The RNA segment that joins what previously had been the tracrRNA (at the 3′ end of the sgRNA) and the crRNA (at the 5′ end) is called a linker loop (at “e”). Everything else is the same (though note that some of the lettering in this diagram is different from that in the previous figure).

CRISPR-Cas9’s power relates to how easy it makes editing the genes of any organism. Before CRISPR-Cas9, other gene editing systems involved designing proteins that would find specific DNA sequences. Engineering these DNA-binding proteins was both expensive and time consuming. By contrast, designing custom-made guide RNA for CRISPR is comparatively quick and easy (you can read this blog post from the Jackson Lab for a comparison of CRISPR with some of the previous techniques).

Paired with sequencing technology (click here to learn how sequencing is done), researchers can identify the sequence for any known gene, and target that gene by creating a custom sgRNA that seeks out the target sequence and makes a double stranded cut.

Figure 7: DNA Repair mechanisms following DNA cutting by CRISPR-Cas9

What happens next depends on the researcher’s goals. What follows lists only a few options, but keep in mind that the technology is changing rapidly.

If, like Dr. He, the goal is to disable a gene, then you allow what’s shown as process number 2 in the diagram to the right. In any cell, a team of enzymes monitors the DNA for double stranded breaks, and then works to recombine the broken DNA by reconnecting the broken strands. The type of repair shown on the left is called non-homologous end joining, a term that only makes sense if you compare it to the system on the right side (number 3).

Any sexually reproducing organism inherits one allele from its mother, and the other from its father. If one allele is damaged, then the second allele can be used as a reference to correct the damaged strand. In non-homologous end joining, the homologous template is not used during the DNA repair process. Rather, a team of enzymes grabs the broken ends and trims or adds nucleotides as needed to reconnect them. This process tends to be inaccurate, and results in deletions or insertions that wind up disabling the gene. In Dr. He’s procedure, this is probably what happened with the twin who had both of her CCR5 receptor genes disabled.

Another DNA repair process is homology directed repair (HDR). In this process, the homologous allele is used as a template by DNA repair enzymes, which repair the double stranded break in a way that leaves the gene in a functional state. Possibly, the second twin in Dr. He’s procedure experienced homologous repair (though it’s also possible that the CRISPR-Cas9 only disabled one of her two alleles).

Note that homologous repair opens up the possibility of adding new DNA. In this method, the targeted DNA breakage by CRISPR-Cas9 is followed by introduction of what’s labeled in figure 7 above as “Donor DNA.” This DNA has ends that are homologous to the broken strand, but with new DNA inserted between the homologous ends. This new DNA could be a working version of a broken gene, or a gene from another species. When homologous repair enzymes repair the broken DNA, they’ll wind up inserting this new DNA. This gives genetic engineers a relatively easy way to create recombinant DNA within living cells. In other words, CRISPR-Cas9 allows genetic engineers to cut genes (by non-homologous end joining, and to paste in new genes (by homology directed repair).

Figure 8: two Cas9s making nicks instead of double stranded cuts

If you want to make sure that your cut will be in the right place (avoiding what’s called “off-target” cleavage), you can use two engineered CRISPR-Cas9s, each of which has been designed to make a single stranded nick in the target DNA (instead of a double stranded cut). These nicks are indicated at 1 and 2 in figure 8, at right. If you add donor DNA (shown at 4) to your cut DNA (at 3), then homologous repair mechanisms will join the DNA together, creating engineered recombinant DNA (5).

In short, a combination of sequencing, CRISPR-Cas9, and DNA repair enable researchers to

  1. Disable genes
  2. Replace disabled genes with edited genes.

Other variations allow CRISPR-Cas9 to be used to visualize genes and to control gene expression.

We’ll look at some applications in a moment. But first, let’s consolidate what you’ve learned.

7. Quiz: CRISPR-Cas9 for Genome Editing

[qwiz random = “true” qrecord_id=”sciencemusicvideosMeister1961-CRISPR 3, Genome Editing (M17)”] [h]

CRISPR-Cas9 as a gene editing system

[i]

[q] In the diagram below, nuclease enzymes are indicated by

[textentry single_char=”true”]

[c*] b

[f] Excellent! Nuclease enzymes are indicated by the scissors at “b.”

[c] Enter word

[f] No.

[c] *

[f] No. Here’s a hint. Nucleases are enzymes that cut DNA. What’s something you cut with?

[q] In the diagram below, the protospacer is indicated by

[textentry single_char=”true”]

[c*] c

[f] Excellent! The protospacer is the target DNA that’s recognized by the crRNA, and it’s indicated by “c.”

[c] Enter word

[f] No.

[c] *

[f] No. Here’s a hint. The protospacer is the DNA that was used to create the spacer, which is the gene for the RNA that complements with (and thus recognizes) DNA sequences from attacking bacteriophage.

[q] In the diagram below, the short DNA sequence that identifies this DNA as viral DNA that should be cut in order to prevent a viral takeover of the cell is

[textentry single_char=”true”]

[c*] d

[f] Excellent! Letter “d” indicates the PAM, the protospacer adjacent motif.

[c] Enter word

[f] No.

[c] *

[f] No. Here’s a hint. You’re looking for a short sequence of DNA that’s immediately adjacent to the protospacer. The protospacer is what’s recognized by “f,” the crRNA.

[q] In the diagram below, tracrRNA is indicated by

[textentry single_char=”true”]

[c*] e

[f] Excellent! Letter “e” indicates tracrRNA, an RNA segment required for activating the CRISPR-Cas9 complex.

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. Here’s a hint. tracrRNA is sometimes described as a handle.

[q] In the diagram below, sg (single guide) RNA is indicated by


[textentry single_char=”true”]

[c*] f

[f] Excellent! Letter “f” indicates sgRNA an engineered RNA segment that links together the crRNA and the tracrRNA in wild-type CRISPR-Cas9 systems.

[c] Enter word

[f] No.

[c] *

[f] No. There’s only one RNA molecule shown above. You can identify RNA because it’s single stranded (as opposed to the double stranded DNA at “a”).

[q multiple_choice=”true”] The CRISPR-Cas9 shown below is

[c*] engineered

[f] Excellent. You know that it’s engineered because there’s only one strand of RNA: an engineered strand that links together tracrRNA and crRNA.

[c] the “wild-type” complex found in species like S.pyogenes.

[f] No. Wild-type CRISPR-Cas9 has two RNA molecules.

 

[q multiple_choice=”true”] In the diagram below, which number is showing homologous repair?

[c] 1

[f] No. This looks more like non-homologous end joining (which often results in loss of function mutations, another clue).

[c*] 2

[f] Excellent. You can see one DNA strand lining up against another one, and the addition of donor DNA. Both are clues that this represents homologous repair.

 

[q] Which number in the diagram below represents a process used to cut targeted sequences of DNA?

[textentry single_char=”true”]

[c*] 1

[f] Nice job. “1” is showing CRISPR-Cas9 cutting a target strand of DNA.

[c] Enter word

[f] No.

[c] *

[f] No. Just look for the part of the diagram where a strand of DNA is being cut into two fragments.

[q] Which number shows the process that a researcher might use to silence or deactivate a gene?

[textentry single_char=”true”]

[c*] 2

[f] Terrific. The deletions or insertions indicated by “2” represent non-homologous end joining repair of DNA. These usually result in mutations that deactivate genes.

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. Just look for the part of the diagram where a strand of DNA is being cut into two fragments.

[q] Which number shows the process that a researcher might use introduce new genes into a cell (or an entire organism).

[textentry single_char=”true”]

[c*] 3

[f] Way to go. Number “3” represents homologous DNA repair accompanied by insertion of donor DNA. This procedure can result of insertion of new DNA organisms into cells.

[c] Enter word

[f] No.

[c] *

[f] No. Just look for the part of the diagram where a strand of DNA is being cut into two fragments.

 

[q] The acronym used to describe the short sequence at “d” is [hangman] . 

[c] PAM

[f] Great!

[q] Letter “c” is a[hangman] . 

[c] protospacer

[f] Correct!

[q] The two Cas9s shown below have been engineered to create a single stranded [hangman] at points 1 and 2, instead of a double stranded cut.

[c] nick

[q] The insertion of a new DNA at steps 3, 4, and 5 below is an example of
[hangman] directed repair.

[c] homology

[/qwiz]

 

 

8. Some CRISPR-Cas9 Applications

Gene editing using CRISPR-Cas9 is progressing as such a pace that by the time you read this the applications listed below will have been superseded by newer ones. Searching for “CRISPR Applications” will get you to the latest developments. In the meanwhile, here’s a starting list of some procedures that biologists are working on…

  1. Potential therapies for inherited genetic disorders.
    1. Duchenne Muscular Dystrophy (DMD) is an X-linked, recessive condition that results in deterioration of muscle tissue, including cardiac (heart) muscle. DMD occurs almost exclusively in boys because girls usually have a second, normal allele on the X chromosome they inherit from their fathers. Boys with this condition rarely live into their thirties. In mice models of DMD, CRISPR was used to target the introns that flank the disease-causing mutation and replace the mutated allele with a functional version, allowing these mice to produce functional muscle tissue (source: Molecular Therapy Nucleic Acids).
    2. Cystic fibrosis is an autosomal recessive condition related to a mutation in a chloride ion channel protein in cell membranes. The mutation results in membrane channels that are absent, defective, or too few in number. Without the channel, chloride doesn’t flow out of cells, which results in uptake of water from the extracellular fluid. In mucus-secreting cells, this causes buildup of a thick, sticky mucus, with various effects in different tissues. In the lungs the result is recurrent bacterial infections. In the digestive tract it results in poor absorption of nutrients.
      In cultured stem cell lines and organoids (3 dimensional tissue cultures used in research) from patients with cystic fibrosis, CRISPR-Cas9 gene-editing combined with homology directed gene repair was used to remove the mutated allele and replace it with a functioning version.  Subsequent experimentation in the repaired organoids showed that the repaired cells were able to process chloride normally (source: Cell: Stem Cell, with an update here).
    3. Sickle Cell Disease is caused by a substitution mutation in the gene for hemoglobin, the oxygen-carrying protein in red blood cells. The mutation results in a single amino acid substitution (with valine inserted in place of glutamic acid). This changes the chemistry of hemoglobin, causing it to polymerize in low oxygen conditions, creating sickle-shaped red blood cells that clog up capillaries, causing tissue damage and severe pain. One proposed gene editing solution that’s nearing clinical trials would involve isolating stem cells from patients with sickle cell disease, using CRISPR-Cas9 to and homology directed repair to remove the mutated allele and replace it with a working version, and then transplanting the corrected cells back into the patient (source: Human Genetics). Another approach involves using CRISPR-Cas9 to create a mutation that increases production of fetal hemoglobin (normally turned off in adults) by downregulating a gene called BCL11A, which suppresses production of fetal hemoglobin after birth.  Turning the fetal hemoglobin gene back on can potentially ameliorate sickle cell disease symptoms in children and adults (source: Hematology Times for an overview, or Molecular Therapy for an in-depth review).
  2. Fighting Viruses: CRISPR-Cas9s evolutionary roots are as a counterattack against bacteriophage . Hence, it’s no  surprise that the system is being used against human viral pathogens. One application being researched is to target HIV when it’s embedded as a provirus in T-cells. Unlike current therapies, which suppress HIV, this therapy could eradicate it from people who are HIV positive (Scientific Reports, volume 8, Article number: 7784 (2018))  Similarly, CRISPR is being investigated as a means to eradicate endogenous retroviruses from pig organs, making them suitable for transplant into humans (sciencemag.org)
  3. Agriculture: CRISPR works with plant cells as well as it works in animal cells. CRISPR based gene editing is already being used to increase pest resistance in crops such as wheat, corn, and tomatoes. In addition, CRISPR is being used to introduce new traits into pennycress, a low value cover crop that’s being engineered for increased biofuel oil production. (source: Nature Reviews Molecular Cell Biology, volume19, pages 275–276 (2018))
  4. Cancer Research and Therapy
    1. In some cancers, Killer T cells that would normally attack tumors are deactivated by signals from cancer cells. A planned trial involves using CRISPR-Cas9 to delete two genes in T cells and then infuse them back into the patients, where they’ll be able to seek out and destroy tumors (source: MIT Technology Review)
    2. The Human Papilloma Virus (HPV) causes genital warts which, if untreated, can progress to cervical cancer. In cervical cancer cell lines (HPV-infected cancer cells that are being grown in cultures), CRISPR-Cas9 targeting of two genes was able to induce these cancerous cells to commit programmed cell suicide, also known as apoptosis. Similar treatments might also be possible for other virally induced cancers, such as Burkitt’s lymphoma, which can be caused by the Epstein Barr Virus (source: Molecular Therapy).

For further Reading about CRISPR and its potential applications (from three of its discoverers), read

The Kavli Prize Interview with Jennifer Doudna, Emmanuelle Charpentier, and Virginijus Šikšnys.

9. CRISPR-Cas9: Cumulative Quiz

The quiz below will test you on your understanding of CRISPR-Cas9 Biology.

[qwiz random = “true” style=”width: 600px !important;” qrecord_id=”sciencemusicvideosMeister1961-CRISPR 4, cumulative quiz (M17)”] [h]

CRISPR-Cas9, Cumulative Quiz

[i]

[q] DNA that originally came from a virus is shown at

[textentry single_char=”true”]

[c*] C

[f] Excellent. The spacer DNA, shown at S1, S2, etc. is how bacteria “remember” previous viral attackers.

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No, but here’s a hint. The DNA of viral origin that’s inserted into the CRISPR array is called a “spacer.”

[q] Spacer DNA is shown at

[textentry single_char=”true”]

[c*] C

[f] Excellent. The spacer DNA, is shown at S1, S2, etc. The spacers are how bacteria “remember” previous viral attackers.

[c] Enter word

[f] No. 

[c] *

[f] No, but here’s a hint. The spacer DNA is interspersed with repeats. 

[q] In the diagram below, a variety of viral DNA fragments are shown at

[textentry single_char=”true”]

[c*] e

[f] Good! Fragmented viral DNA is shown at “e.”

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The DNA inserted by the virus is shown at “c.” If that DNA were fragmented, what would it look like? 

[q] In the diagram below, the viral DNA that will form the new spacer is shown at

[textentry single_char=”true”]

[c*] f

[f] Good! The viral DNA fragment that will become a new spacer is shown at “f.” 

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. In the diagram, find the DNA fragment that’s going to be inserted to the CRISPR array (at letter “G”). 

[q] In the diagram below, the CRISPR array is shown at

[textentry single_char=”true”]

[c*] j

[f] Correct! The CRISPR array consists of spacers (i) and repeats (h).

[c] Enter word

[f] Sorry, that’s not correct.

[c] *

[f] No, but here’s a hint. The CRISPR array consists of spacers (i) and repeats (h). See if you can connect the diagram below to the one above. 

[q] In the diagram below, the only letter that points to the Cas9 protein before it has associated with RNA is

[textentry single_char=”true”]

[c*] J

[f] Excellent! “J” shows the Cas9 protein before it has associated with any RNAs. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. Letter “o” shows CRISPR-Cas9. Where can you find the Cas9 without any RNA?

[q] In the diagram below, pre-CRISPR RNA is shown at

[textentry single_char=”true”]

[c*] g

[f] Awesome! Letter “g” shows pre-CRISPR RNA.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. Letter “f” shows CRISPR DNA.

[q] In the diagram below, crRNA is shown at

[textentry single_char=”true”]

[c*] p

[f] Awesome! Letter “p” shows crRNA. You can tell because it’s binding with viral DNA. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. Look for single stranded RNA that’s binding with viral DNA. 

[q] In the diagram below, viral DNA that’s been cleaved by Cas9 is shown at

[textentry single_char=”true”]

[c*] q

[f] Awesome! Letter “q” shows cleaved viral DNA. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. Viral DNA is going to be fragmented after it’s acted upon by CRISPR-Cas9.

[q] In the diagram below, mature CRISPR-Cas9 is shown at

[textentry single_char=”true”]

[c*] o

[f] Awesome! Letter “o” shows mature CRISPR-Cas9

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. CRISPR-Cas9, once mature, can find and fragment viral DNA with a protospacer that complements its crRNA.

[q] In the diagram below, the protospacer would be found within

[textentry single_char=”true”]

[c*] c

[f] Awesome! Letter “c” shows viral DNA. Within that DNA sequence is a protospacer that CRISPR-Cas9 can bind with and cleave apart. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The protospacer is a sequence within the viral DNA. Find the viral DNA, and you’ll see where the protospacer has to be. 

[q] In the diagram below, the CRISPR array is found at

[textentry single_char=”true”]

[c*] c

[f] Awesome! Letter “c” shows the CRISPR array.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. You’re looking for an array of repeats and spacers. 

[q] In the diagram below, spacers are indicated by which letter? (Note: don’t worry about the subscript)

[textentry single_char=”true”]

[c*] b

[f] Awesome! Letter “b” indicates the spacers.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The spacers are part of the CRISPR array, and they’re separated by identical repeat sequences. 

[q] In the diagram below, the bacterial chromosome is indicated by

[textentry single_char=”true”]

[c*] d

[f] Awesome! Letter “d” indicates the bacterial chromosome. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The bacterial chromosome is a circular piece of DNA that includes the CRISPR array, CRISPR associated genes, and all of the bacterial cell’s other genes. 

[q] In the diagram below, pre-crRNA is indicated by

[textentry single_char=”true”]

[c*] e

[f] Awesome! Letter “e” indicates pre-crRNA. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. pre-crRNA will be transcribed from the CRISPR DNA, which is shown at “c.”

[q] In the diagram below, crRNA that hasn’t yet associated with the Cas9 protein is shown at

[textentry single_char=”true”]

[c*] f

[f] Awesome! Letter “f” indicates crRNA. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. At stage 3, you can see four CRISPR-Cas9s. Each of which consists of a Cas9 protein (in blue) associated with a crRNA.

[q] In the diagram below, a protospacer is shown at which letter? Note: don’t worry about using a subscript in your answer.

[textentry single_char=”true”]

[c*] h

[f] Awesome! Letter “h” indicates the protospacer. This is the viral DNA sequence that complements the crRNA, and which is transcribed from the spacer. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The protospacer is viral DNA that’s complementary to crRNA.

[q] In the diagram below, the PAM is shown by which letter? Note: don’t worry about using a subscript in your answer.

[textentry single_char=”true”]

[c*] i

[f] Awesome! Letter “i” indicates the PAM: the protospacer adjacent motif.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The PAM is an acronym for protospacer adjacent motif. It’s adjacent to the protospacer, which is a recognized sequence within the viral DNA.

[q] In the diagram below, the PAM is shown by which letter?

Courtesy of Tebu-Bio (www.tebu-bio.com)

[textentry single_char=”true”]

[c*] b

[f] Awesome! Letter “i” indicates the PAM: the protospacer adjacent motif.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The PAM is an acronym for protospacer adjacent motif. It’s adjacent to the protospacer, which is a recognized sequence within the viral DNA.

[q] In the diagram below, which short sequence of DNA bases acts a signal to Cas9 saying “it’s ok to cleave this DNA.”

Courtesy of Tebu-Bio (www.tebu-bio.com)

[textentry single_char=”true”]

[c*] b

[f] Awesome! Letter “i” indicates the PAM: the protospacer adjacent motif.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. You’re looking for the protospacer adjacent motif, or PAM. Find something that’s adjacent to the protospacer.

[q] In the diagram below, the sgRNA is indicated by letter

Courtesy of Tebu-Bio (www.tebu-bio.com)

[textentry single_char=”true”]

[c*] a

[f] Awesome! Letter “a” indicates the sgRNA, an engineered RNA that fuses together crRNA and tracrRNA.

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. RNA is single stranded.

[q] In the diagram below, the protospacer is indicated by letter

Courtesy of Tebu-Bio (www.tebu-bio.com)

[textentry single_char=”true”]

[c*] c

[f] Awesome! Letter “c” indicates the protospacer, the sequence of DNA that CRISPR-Cas9 has been programmed to seek out and cleave. 

[c] Enter word

[f] No.

[c] *

[f] No, but here’s a hint. The protospacer is the sequence of DNA that CRISPR-Cas9 has been programmed to seek out and cleave. 

[q] In the diagram below, non-homologous end joining is indicated by letter

[textentry single_char=”true”]

[c*] d

[f] Terrific! Letter “d” indicates non-homologous end joining. 

[c] Enter word

[f] No.

[c] *

[f] No. Look for a part of the diagram in which the ends of the DNA are being stitched together, without reference to any other strand of DNA. 

[q] In the diagram below, which letter indicates a process that could be used to deactivate/knock out a gene.

Courtesy of Tebu-Bio (www.tebu-bio.com)

[textentry single_char=”true”]

[c*] d

[f] Terrific! Letter “d” indicates non-homologous end joining. The mutations that are associated with that process usually wind up deactivating or knocking out gene activity. 

[c] Enter word

[f] No.

[c] *

[f] No. Look for a part of the diagram in which the ends of the DNA are being stitched together, without reference to any other strand of DNA. 

[q] In the diagram below, which letter indicates a process that could be used to insert a new gene.

Courtesy of Tebu Bio (www.tebubio.com)

[textentry single_char=”true”]

[c*] e

[f] Terrific! Letter “e” indicates homology directed repair. The part of the sequence indicated by the red rectangle indicates insertion of new DNA. 

[c] Enter word

[f] No.

[c] *

[f] No. Look for a part of the diagram in which the DNA is repaired by using a homologous template as a reference. 

[/qwiz]

10. Links

At least for now, this tutorial ends this module on biotechnology and genetic engineering. If you have ideas for other topics, please email me. Otherwise, here’s a link back to the Genetic Engineering and Biotechnology Main Menu. Use the links above for other biology tutorials.