CRISPR-Cas9

CRISPR-Cas9 is a technical innovation that is revolutionizing molecular biology. It hasn’t found its way into the Bio 6B lab (yet!), but it uses some of the same principles that underlie other common techniques in molecular biology, and it relates to some of the concepts of Bio 6B. 

What does CRISPR-Cas do?

CRISPR-Cas is a system for cutting and editing the nucleotide sequences of DNA molecules. Originally a naturally occurring antiviral defense system in bacteria, it has been adapted for a wide range of laboratory goals that require recognizing and altering specific nucleotide sequences in DNA.

Start with some  videos

CRISPR Explained. Mayo Clinic; 2 minutes. A clear, simple overview to the potential use of CRISPR-Cas9 in medicine.

CRISPR: Gene editing and beyond. Nature; 4 minutes. A more detailed description of CRISPR mechanisms and variations. Outstanding animation.

Biological Background

Like most molecular biology techniques, CRISPR isn’t entirely a human invention; it’s based on naturally occurring biological processes that have been repurposed for lab use. It makes sense to examine the original biology of CRISPR before looking at its technological applications.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The term refers to a distinctive set of nucleotide sequences that is found in many bacterial genomes and is involved in antiviral defense. Like all other organisms, bacteria are constantly under attack by viruses, known as bacteriophages (or phages for short). The CRISPR-Cas9 system acts like an immune system, enabling bacterial cells to recognize viral DNA sequences and cut up the viral DNA, preventing the virus from destroying the cell. This diagram shows the basic steps:

Image credit: BioRender.

Phase 1: Acquiring viral DNA

When a bacteriophage infects a cell, the phage DNA is injected into the cell. If the viral infection is successful, the viral genes can be transcribed, producing viral proteins and starting the process of viral replication, which leads to the destruction of the cell. However, Streptococcus and other bacteria have a defense system that starts with acquiring viral DNA sequences. First, Cas1 and Cas2 enzymes cut the viral DNA into fragments. Then the fragments of viral DNA are inserted into the bacterial chromosome at the CRISPR locus. 

In this diagram, the spacers are fragments of viral DNA taken from various viruses. Between the spacers are short stretches of repeated nucleotide sequences (shown in gray). These repeats happen to be palindromic, meaning that their nucleotide sequences read the same in both directions. The repeats between the spacers are short, and they are clustered together at the CRISPR locus. This gives rise to the name CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats. This name was given to the sequences when they were first discovered, before researchers discovered that CRISPR functions like a bacterial immune system.

Phase 2: Defense

Once a bacterial cell has incorporated viral DNA into its own genome, the viral nucleotide sequences can be used to recognize the same type of phage if it infects the cell again. 

  • First, parts of the CRISPR locus are transcribed, including the virus-derived spacer and the bacteria’s own palindromic repeats. These RNA transcripts go through multi-step processing to form a guide RNA (I’ll skip some of the RNA-processing steps here). 
  • The guide RNA (or gRNA) forms a complex with the Cas9 protein. The spacer region of the RNA matches a virus-derived nucleotide sequence, while the bacterial palindromic repeat portion of the RNA interacts with the Cas9 protein.
  • The gRNA binds to viral DNA where it finds matching complementary sequences. This process is called hybridization; it’s similar to a PCR primer annealing with its target sequence. With the gRNA in place, the Cas9 protein can act on the viral DNA. Cas9 is an endonuclease: an enzyme that cuts DNA. The gRNA determines the location, and the Cas9 protein catalyzes the cutting of both strands of viral DNA.
  • When Cas9 cuts up the viral DNA, the cell is protected from the virus.

The end result is that, if a bacterial cell manages to survive its initial infection with a phage, the cell can “learn” to recognize that phage in the future and quickly fight off new infections. Since the viral nucleotide sequences are inserted into the cell’s chromosomal DNA, the information will be passed on when the cell divides. 

CRISPR-Cas9 compared to restriction enzymes

Cas9 is an endonuclease: an enzyme that cuts DNA. Other endonucleases, called restriction endonucleases (or restriction enzymes), are commonly used in molecular biology labs, including the 6B lab, in experiments that involve cutting and joining (ligating) DNA. Restriction enzymes are proteins that bind to specific nucleotide sequences, often six nucleotides long, and cut both strands of DNA wherever that sequence occurs. Restriction enzymes exist in bacteria, and they can help protect bacterial cells against viral DNA, but they are limited by their all-protein structure. Viral genomes evolve very rapidly, and it’s not possible for the restriction enzyme genes to evolve rapidly enough to recognize the changing targets in viral DNA. In addition, since restriction enzymes recognize only very short nucleotide sequences, their ability to recognize specific genetic features will always be limited.

The CRISPR-Cas system extends the endonuclease concept by using an RNA molecule instead of a protein to recognize the DNA nucleotide sequence that will be cut. The RNA, called a guide RNA, specifically base pairs with a viral DNA sequence (typically around 20 nucleotides long), making this system more specific and adaptable than restriction enzymes. Bacterial genomes commonly contain multiple virus-derived fragments of DNA (called spacers), that can be transcribed to produce guide RNAs that recognize specific phages. This RNA-protein system is much more adaptable than all-protein restriction enzymes, because the same enzyme can be used with an unlimited range of RNA sequences to target any DNA nucleotide sequence.

When this system was first discovered, biologists recognized that it could function as a sort of bacterial immune system. It’s similar to the human immune system, but it recognizes nucleotide sequences instead of protein antigens. This was an amazing insight, revealing how bacteria can adapt to the constant onslaught from bacteriophages (which are probably the most abundant biological entities on earth). Many researchers were also quick to recognize that the CRISPR-Cas9 system could be put to other uses. Jennifer Doudna and Emmanuele Charpentier were awarded the 2020 Nobel Prize in Chemistry for their role in the discovery and development of CRISPR (see the links at the bottom of this page for more information).

Gene editing with CRISPR-Cas9

Molecular biologists are excited about CRISPR-Cas9 because it can potentially enable them to generate any desired changes in the genome of an organism. While this sort of gene editing has been done with other methods, the CRISPR-Cas9 streamlines the process, opening new avenues of investigation. The heart of the CRISPR-Cas9 system is the ability to cut DNA at a specific location, anywhere in the genome.

Image credit: BioRender.

If a CRISPR-Cas9 system, with the appropriate RNA and enzyme components, is introduced into a eukaryotic cell, the gRNA can base pair with a matching sequence in the chromosomal DNA and allow the Cas9 enzyme to cut both strands of DNA at a specific location. 

If the goal is to edit a gene, then creating this specific cut is half of what needs to be done. The essential next step is to introduce desired sequence changes in the chromosomal DNA. For this, molecular biologists make use of the cell’s natural DNA repair processes, in two fundamentally different ways.

Non-homologous end joining

The double-stranded break introduced by Cas9 will activate the cell’s DNA repair system, which repairs the break. One DNA repair mechanism is called non-homologous end joining; it simply attempts to join the cut ends of the DNA back together. However, this repair system is error-prone; in many cases, extra nucleotides are inserted or deleted at the repair site. If this insertion or deletion occurs in the coding sequence of a gene, it can create a frameshift, disabling the gene. 

This imperfect repair system turns out to be useful; researchers can use it to create targeted “knockouts,” disabling specific genes. This is important because, although modern sequencing technology has provided biologists with vast amounts of genomic data, nobody actually knows exactly what many of the genes do. By creating a knockout strain of fruit fly, or mouse, or other organism, lacking a functional copy of a specific gene, researchers can investigate the function of the gene.

Non-homologous end joining is a somewhat blunt tool, since researchers don’t have precise control over the final nucleotide sequence in the chromosome. As it turns out, living cells also provide another, more controlled mechanism of DNA repair.

Homology-directed repair

One of the advantages of being diploid is that diploid cells are more tolerant of DNA damage than haploid cells. One reason for this is simply that diploid cells have two copies of each locus; if one copy is mutated, the other copy can be used as a backup. In addition, diploid cells can also use the undamaged DNA of one copy of a chromosome as a template to repair the damaged DNA in the homologous chromosome. (In the diagram above, the other chromosome would act as the “donor template.”) This process is known as homology-directed repair. In short, if a chromosome is missing a piece, the appropriate nucleotide sequence can be copied from the homologous chromosome. (This wouldn’t work in haploid cells, because they don’t have homologous chromosomes.)

Homology-directed repair is essential for the long-term survival of eukaryotes, especially for long-lived multicellular eukaryotes such as ourselves. The DNA repair processes help protect us against cancer. Two well-known genes involved in homology-directed repair processes are BRCA1 and BRCA2, which were first identified because they are tumor suppressors that play important roles in specific types of cancer. These genes encode proteins that are needed for DNA repair; cells with loss-of-function mutations in these loci are at greater risk of becoming cancerous.

The process of homology-directed repair can also be put to use in gene editing with CRISPR-Cas9. In the diagram above, the “donor template” is a piece of DNA added by researchers to serve as a template for repairing the DNA break. The donor template must be carefully designed to match the cut ends of the DNA and contain the desired nucleotide sequences to be inserted into the chromosome. With a careful choice of guide RNA and donor template, CRISPR-Cas9 can be used to make almost any desired change in the nucleotide sequence of a chromosome.

Other CRISPR techniques

The techniques I’ve described so far are based on a three part-process: the guide RNA recognizes a specific nucleotide sequence in the DNA, Cas9 cuts the DNA at that site, and then the DNA is repaired, introducing a mutation. Researchers have also developed numerous other approaches based on CRISPR-Cas9. Mutant versions of the Cas9 protein have been developed in which the DNA-cutting “scissors” activity is disabled, and other molecules are attached to the Cas9 protein. The guide RNA-Cas9 complex binds to a specific nucleotide sequence, carrying other functionally important molecules to a specific site on the chromosome, as shown in this diagram:

Image credit: Nik Spencer/Nature.

As this diagram shows, various engineered versions of Cas9 have been developed, making it possible to use this system for regulating gene expression without changing the nucleotide sequences of the DNA.

The future of CRISPR

CRISPR-Cas9 has received a great deal of attention both in the scientific world and in the popular press, because it holds the possibility of making any kind of genetic change in any organism. Much of the attention has focused on one basic idea: genetically engineered humans. With a new gene editing toolkit available, will it soon become normal for all of us to make all sorts of changes in our own genomes? Will we now be able to give ourselves mutant superpowers, or simply remake ourselves to fit some desired ideal? In short, no. There are several reasons why this isn’t going to happen soon.

Most traits aren’t simple

You can’t give yourself a gene for superhuman intelligence, because there is no such gene. Complex traits like intelligence are affected by hundreds of genes as well as by numerous non-genetic factors, all interacting in ways that may never be understood. In fact, very few traits are simply controlled by a single locus. 

CRISPR isn’t that easy

CRISPR-Cas9 applications aren’t as easy to achieve as the simple diagrams on this page might make it seem. Eukaryotic genomes are complex, and cells contain multiple mechanisms that normally take charge of genome integrity. For example, the p53-mediated DNA repair pathways may fight against experimental modifications with CRISPR-Cas9. Even a well-designed experiment could potentially generate unplanned changes in the genome. The technology is still in its developmental stages.

Somatic vs. germline modifications 

The invention of CRISPR-Cas9 doesn’t mean that it has suddenly become possible to edit the genome of every cell in your body. It will always be a challenge to introduce the RNA and enzymes into the desired cells. As with RNA vaccines, a system is needed to allow large, charged molecules of RNA and protein to cross cell membranes. These systems can’t deliver CRISPR-Cas9 components to every cell. Many applications of CRISPR-Cas9 involve editing a small number of cells, in a targeted way. Some of the most promising medical applications involve targeting specific cell types, such as erythropoietic (blood) stem cells, which can be modified outside the body and then re-inserted into the body. 

Approaches like this are called somatic cell modifications; they edit the genomes of some cells in the body, but they don’t alter the germline (egg or sperm) cells. Germline modifications would be passed to the next generation, but somatic modifications won’t.

Moratorium on human germline modification

A lot of things could go wrong with genetically modifying humans. There is a lot that isn’t known, both about how CRISPR-Cas9 works in eukaryotic cells and about genome functions in general. For this reason, the world’s major scientific organizations have, by consensus, agreed on a moratorium on human germline modification. Responsible scientists don’t think human germline modification is a good idea at this point.

Germline editing of human DNA by CRISPR-Cas9 or other means might someday become common, or it might not. A great many practical and ethical questions remain to be answered before that comes to pass. For the near future, the most important value of this new toolkit is likely to be the fundamental knowledge that is gained through experiments that don’t involve genetically engineered humans. The research applications of CRISPR-Cas9 are growing by the day. From basic research on gene function to new diagnostic methods for viruses, this technology is being put to new and unexpected uses. Before we can successfully edit genomes, we need to understand them, and CRISPR-Cas9 provides a powerful new approach to gaining that understanding.

Review

Terms & concepts

  • Bacteriophage (also called phage): any virus that infects and potentially kills bacteria.
  • Cas. There are multiple Cas (CRISPR-associated) enzymes. Some, like Cas1 and Cas2 in the diagram on this page, function in inserting viral DNA into the bacterial genome. Cas9 cuts viral DNA (or any other DNA), with guidance from an RNA molecule.
  • CRISPR. On a quiz, I’ll ask you how it works, rather than what it stands for.
  • Guide RNA (gRNA). The RNA components in CRISPR-Cas9 include a segment that hybridizes with target DNA and segments that interact with the Cas9 protein. If you look at various CRISPR-Cas9 resources, you’ll see various terms for  these specific pieces of RNA, but you don’t need to remember those terms for this class. In lab procedures, researchers typically combine several RNA functions into one short RNA, called the guide RNA (gRNA) or sometimes called the single guide RNA (sgRNA).
  • Homology-directed repair
  • Hybridization: this term refers to any situation in which two different nucleotide sequences base pair with each other. Guide RNA binding in CRISPR-Cas9 and primer binding in PCR are examples of hybridization; there are numerous other examples in other laboratory procedures. These nucleic acid processes are completely separate from the other meaning of hybridization, which is when individuals of two different species mate and produce offspring.
  • Non-homologous end joining

Review Questions

  1. Restriction enzymes are endonucleases that cut DNA at specific nucleotide sequences. Restriction enzymes have been used in labs for decades as tools for cutting DNA and creating recombinant DNA molecules. How is CRISPR-Cas9 similar to a restriction enzyme, and how is it different? Why is the CRISPR-Cas9 system such a revolutionary advance in molecular biology?
  2. What do non-homologous end joining and homology-directed repair normally do in cells? How are they put to use in CRISPR-Cas9?
  3. Bacterial cells use specific RNA sequences to recognize bacteriophage DNA. How do the bacterial cells acquire these RNA sequences? 
  4. In some CRISPR-Cas9 experiments, researchers use modified versions of Cas9 that lack endonuclease activity. What are these modified Cas9 enzymes used for?
  5. Why would non-homologous end joining in CRISPR-Cas9 be likely to create a knockout mutation?

References & further reading

History and ethics of CRISPR-Cas9

CRISPR-Cas9 is one of the most remarkable discoveries and technological developments of our time. Two of the key discoverers, Jennifer Doudna and Emmanuele Charpentier, were awarded the Nobel Prize in Chemistry in 2020 for their contributions. Dr. Doudna, of UC Berkeley, has also become the most prominent voice in speaking about the ethical implications of gene editing technology.

Pioneers of revolutionary CRISPR gene editing win chemistry Nobel. Nature.

Genetic scissors: a tool for rewriting the code of life from the Nobel Prize website.

Crispr-Cas9 explained: the biggest revolution in gene editing. 4-minute, non-technical video from The Guardian, featuring Jennifer Doudna talking about the implications of gene editing both in terms of technological possibilities and ethical questions.

The ethics of CRISPR gene editing with Jennifer Doudna. 2-minute video from UC Berkeley News; this video goes along with CRISPR-Cas9 gene editing and how it works - with Jennifer Doudna.

Perspectives on gene editing. Harvard Gazette, 2019. Summarizes key ethical issues related to gene editing.

DNA repair

What happens when your DNA is damaged?. Monica Menesini, TED-Ed. 5-minute video with clear and simple animations. Explains how homologous recombination and non-homologous end joining fit into normal cell biology.

CRISPR-Cas9 techniques & applications

The expanding CRISPR toolbox. Nature & Dharmacon. A poster (pdf) showing various ways that CRISPR-Cas9 is being used in the lab for both genetic and epigenetic manipulation.

CRISPR: gene editing is just the beginning. The real power of the biological tool lies in exploring how genomes work. Nature, 2016. Includes the “Hacking CRISPR” diagram shown above.

CRISPR-Based Anti-Viral Therapy Could One Day Foil the Flu—and COVID-19. Collins 2021, NIH. This article describes an approach that brings CRISPR full-circle: the process that evolved as a virus-fighting mechanism in bacteria could be repurposed as a virus-fighting mechanism in humans. This doesn’t involve editing the human genome; instead, it uses CRISPR guide RNA to target viral RNA, with an RNA-cutting enzyme attached. For more on this approach, see Could Crispr Be Humanity's Next Virus Killer? (Wired).

CRISPR gene therapy shows promise against blood diseases. Nature, 2020.

Sickle Cell Gene Therapy Using CRISPR. From Synthego, a biotech company that is developing several CRISPR-based therapeutics for human use.

After the Nobel, what next for Crispr gene-editing therapies? Ball, 2021. A news article from the Guardian.

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