CRISPR-Cas Gene Editing Teaching Resources
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|A Microbial Immune System||A Gene Editing System||Applications of CRISPR Technology||References|
On This Page
|A Microbial Immune System||A Gene Editing System||Applications of CRISPR Technology||References||CRISPR for the Classroom|
One of the most exciting recent developments in genetic engineering is CRISPR-Cas9 (CRISPR). CRISPR derives its name from "clustered regularly interspaced short palindromic repeats," genomic sequences that microbes use to defend themselves against viral attacks. Along with CRISPR associated (Cas) proteins, bacteria use the sequences to recognize and disarm future invading viruses. Scientists have adopted this system for use in genetic engineering.
CRISPR-Cas technology allows scientists to edit genes and manipulate gene expression with a level of ease that was not possible using other methods. Importantly, it also allows researchers to edit genes within living organisms, a fact that supports the use of CRISPR-Cas in a far-reaching range of applications from basic research to the development of novel therapies and other biotechnology products.
CRISPR-Cas — A Microbial Immune System
Humans have complex immune systems that involve the coordinated activities of multiple cell types, organs, and signaling systems to recognize and respond to active infections. Prokaryotes — bacteria and archaea — also have a form of adaptive immunity that allows them to recognize and respond to viral infections.
Some prokaryotic genomes contain short, palindromic DNA sequences that are repeated many times, with unique "spacer" sequences between the repeats. These "clustered regularly interspaced short palindromic repeats" (CRISPR) are followed by short segments of spacer DNA that match DNA sequences found in bacteriophage genomes (bacteriophage are viruses known to infect bacteria). Groupings of CRISPR-associated (Cas) genes are also found next to CRISPR sequences, and these Cas genes encode enzymes that cut DNA in specific places, like precise molecular scissors.
CRISPR-Cas9 sequences work together to provide an immune response in which a microbe could recognize invading DNA and unleash Cas enzymes to cut and disarm it. CRISPR sequences and the Cas enzymes are the keys to the microbial "adaptive immune response," which involves three phases:
- Cutting and Capture — When bacteria are infected by a virus, they use their CRISPR system to cut up the invading viral DNA and insert pieces of it (spacers) into their own genome as a "memory" of the infection.
- Monitoring — Bacteria transcribe the spacers into RNA, which can form a complex with the Cas9 enzyme. These complexes monitor the cell for any DNA sequence complementary to the RNA.
- Defense — If matching (viral) DNA is encountered, the spacer RNA-Cas9 complex binds to it and cuts the viral DNA to prevent it from replicating. This halts the viral infection.
Using this system, bacteria can collect sequences from many different infecting viruses to create a "library." Since the CRISPR sequence is contained in genomic DNA, it is passed on to each generation, and the library continues to change and adapt to more common threats over time.
Acquiring Foreign DNA Sequences Defend Against Future Infection
CRISPR-Cas9 — A Gene Editing System
The CRISPR-Cas9 system uses modified components of the bacterial CRISPR system to direct target-specific cutting of double-stranded. DNA repair mechanisms then take over to fix the break in a manner that modifies the genetic sequence that has been cut.
Cutting the DNA
- Cas9 enzyme (Cas9) — an endonuclease that cuts both strands of DNA at a specific site. Multiple types of Cas enzymes are found in nature, but Cas9 is commonly used in the laboratory
- Single guide RNA (sgRNA) — an engineered RNA that forms a complex with Cas9. The sgRNA is a fusion of two regions that occur as separate RNAs in nature:
- Programmable region — part of the CRISPR RNA or crRNA in nature, a 20-nucleotide region that is complementary to the target region and defines the target DNA sequence that Cas9 cuts. Scientists customize this sequence for their own targets
- Scaffold region — the trans-activating CRISPR RNA or tracrRNA in nature, this region forms a multi-hairpin loop structure (scaffold) that binds in a crevice of the Cas9 protein
- Protospacer adjacent motif (PAM) — required for Cas9 function, this sequence motif is immediately downstream of the target sequence. Cas9 recognizes the PAM sequence 5’-NGG where N can be any nucleotide (A, T, C, or G). When Cas9 binds the PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA
5 Steps of Cas9 DNA Cleavage
1. Cas9 Binds an sgRNA
Cas9 recognizes and binds the scaffold (tracrRNA) region of a sgRNA. The nucleotide sequence of the scaffold region determines its structure, which is tailored to fit within the Cas9 protein as a key fits into a lock.
2. The Cas9-sgRNA complex binds to a PAM site on the target DNA
Cas9 requires a particular PAM sequence (5’-NGG) to be present directly adjacent to the protospacer sequence. When the Cas9-sgRNA complex recognizes and binds a PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA.
3. The guiding region of the sgRNA binds to the target DNA sequence
The programmable region of the sgRNA attempts to base-pair with the DNA. If a match is found, the process continues. Otherwise, the complex releases and attempts to bind another PAM and target DNA sequence.
4. Cas9 makes a double-stranded break in the DNA three base pairs upstream of the PAM
5. The complex releases from the DNA
The Cas9-sgRNA complex releases the cut DNA and is ready to repeat the process.
Repairing the Break to Engineer the Change
Researchers can use the cell’s own DNA repair machinery to modify, insert, or delete a nucleotide sequence. The repair can happen in two ways:
- Non-homologous end joining (NHEJ) — enzymes reconnect the ends of the double-stranded break back together. This process may randomly insert or delete one or more bases and can cause mutations that can disrupt gene function or expression
- Homology directed repair (HDR) — proteins patch the break using donor template DNA. Researchers design the donor template DNA that may include a desired sequence flanked on both sides by "homology arms" that match the sequence upstream and downstream of the cut. A complementary DNA strand is created during the repair
Applications of CRISPR Technology
With CRISPR, targeted disruption of any gene — in most organisms — is possible. It allows scientists to modify genomic DNA with precision to ensure that no other genes or sequences are unintentionally disrupted. CRISPR technology is easier, faster, and less expensive than other gene-editing techniques and can be used to edit multiple genes at the same time in a single cell. Finally, CRISPR requires the introduction of only one protein (Cas9) and one sgRNA into a cell. Such a powerful technology can be expected to have a vast range of applications.
Researchers are looking to CRISPR as a technique for editing out genetic defects that result in sickle cell disease, cystic fibrosis, hemophilia, and muscular dystrophy, and for developing more targeted and effective cancer treatments. One study showed that adult rats engineered to have a genetic form of blindness could be treated using CRISPR gene therapy (Berry et al. 2019). The goal is to someday have patients' diseased cells removed, "fixed" with CRISPR, and then returned to their bodies to treat various conditions or have diseased organs be treated directly with CRISPR. The potential for using CRISPR to change genetic traits in humans has raised serious concerns, about possible unintended effects, as well as ethical questions. The ease of applying CRISPR has caused worry about the potential misuse of the technology. Despite these concerns, CRISPR is revolutionizing many aspects of biotechnology and scientific research.
CRISPR technology is expected to accelerate the development of new, improved crops. The technology has produced crops and livestock with desirable traits such as faster growth, higher nutrient content, and disease resistance. And, since CRISPR technology can modify genes without introducing new genes, CRISPR-modified plants may be subjected to lighter regulations than other genetically modified crops.
Scientists have used a modified CRISPR-Cas9 system to create a yeast strain to produce lipids and polymers. These molecules could be useful in the development of biofuels, adhesives, and fragrances. Currently, these lipids and polymers are made synthetically from non-renewable petroleum-based materials that are more expensive and could present safety risks.
Scientists are experimenting with using CRISPR to engineer "gene drives" to spread specific genes through a population of insect pests that cause them to die or become infertile. This technique is being considered to eradicate mosquitoes carrying human pathogens like malaria parasites or Zika virus.
Using CRISPR in your research? See Bio-Rad’s Solutions for Gene Editing Workflows
Gene Editing and CRISPR References
- Berry MH et al. (2019) Restoration of high-sensitivity and adapting vision with a cone opsin. Nature Communications 10, 1221.
- Carroll D (2017) Genome editing: past, present, and future. Yale J Biol Med 90, 653–659.
- Cong L et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823.
- Cribbs A et al. (2017) Science and bioethics of CRISPR-Cas9 gene editing: an analysis towards separating facts and fiction. Yale J Biol Med 90, 625–634.
- Jinek M et al. (2012) A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821.
- Park A (2016) A new technique that lets scientists edit DNA is transforming science — and raising difficult questions. Time 43–48.
CRISPR for the Classroom
Out of the Blue CRISPR Kits
Use CRISPR-Cas9 gene editing technology for editing the lacZ gene in E. coli. Visualize results with blue-white screening and confirm them by PCR. Deepen your understanding of CRISPR, its promise and risks, and the ethics behind its many applications.
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