CRISPR-Cas9 Genome Editing Technology

Summary

The script explores the groundbreaking CRISPR-Cas9 genome editing technology, detailing its origin from bacteria's adaptive immunity to a revolutionary tool in molecular biology. It explains the mechanism involving CRISPR arrays, tracrRNA, and Cas9 protein, leading to precise gene editing. The summary also touches on the technology's applications in areas like cancer immunotherapy, AIDS treatment, and detecting SARS-CoV-2, highlighting its potential to cure diseases and improve crops, while acknowledging ethical considerations.

Takeaways

• 🧬 The CRISPR-Cas9 system is a revolutionary genome-editing technology that allows for precise and site-specific gene editing.
• 🔬 CRISPR was first discovered in 1987 by Atsuo Nakata and his team in the Escherichia coli genome, and it was later understood to be a part of prokaryotic adaptive immunity.
• 🛡️ CRISPR functions as a defense mechanism in bacteria by integrating foreign DNA from viruses or mobile genetic elements into its genome to recognize and neutralize future infections.
• 🧬🧬 The CRISPR array is composed of palindromic repeats and spacers derived from foreign DNA, which the bacteria use to remember past infections.
• 🌟 The CRISPR-Cas9 system was adapted for use in eukaryotic species, including humans, by Jennifer Doudna and Emmanuelle Charpentier, who were awarded the Nobel Prize in Chemistry in 2020.
• 🧪 The CRISPR-Cas9 system uses a single guide RNA (sgRNA) fused with Cas9 protein to target and cleave specific DNA sequences in a process that can be controlled in the lab.
• 🧵 DNA repair after CRISPR-Cas9 cleavage can occur through two pathways: non-homologous end joining (NHEJ), which can introduce indels, or homology-directed repair (HDR), which uses a DNA template and is less error-prone.
• 💉 CRISPR has significant applications in medicine, including cancer immunotherapy where T cells are genetically modified to better recognize and kill cancer cells.
• 🛡️🦠 CRISPR technology is also being used to develop treatments for HIV/AIDS by targeting the proviral form of the virus within immune cells.
• 🌐 The technology has been instrumental in developing assays for detecting SARS-CoV-2, aiding in the global response to the COVID-19 pandemic.
• 🌱 In agriculture, CRISPR is used to create crops with improved resistance to diseases, enhanced phenotypes, and increased yields, potentially revolutionizing food production.

Q & A

What is CRISPR-Cas9 and why is it significant in biotechnology?

CRISPR-Cas9 is a genome editing technology that allows for precise and site-specific gene editing, providing an unprecedented level of control in manipulating the genetic information of a living organism.

Who first reported the presence of CRISPR and in what organism?

Atsuo Nakata and his team from Osaka University first reported the presence of CRISPR in the Escherichia coli genome in 1987.

What are the CRISPR sequences and why are they described as palindromic?

CRISPR sequences are short, repeated sequences of DNA nucleotides found within the genome of prokaryotes. They are described as palindromic because they read the same from 5' to 3' on one strand of DNA and from 5' to 3' on the complementary strand.

What role does CRISPR play in prokaryotic adaptive immunity?

CRISPR is a key component of the adaptive immunity in prokaryotes, protecting them from attacks by viral DNA, bacteriophages, and plasmids by incorporating small pieces of foreign DNA into the CRISPR locus to generate a memory of past infections.

What is the function of Cas9 in the CRISPR system?

Cas9 is a nuclease enzyme that cleaves DNA at specific nucleotide linkages, acting like a pair of scissors. It forms complexes with crRNA and tracrRNA to recognize and cut viral DNA sequences, neutralizing the virus.

How was CRISPR adapted for genome editing in humans and other organisms?

In 2012, Jennifer Doudna and Emmanuelle Charpentier proposed using the CRISPR-Cas9 system as a programmable toolkit for genome editing. They combined crRNA and tracrRNA into a single guide RNA (sgRNA), enabling precise DNA targeting and cleavage in various species.

What are the two DNA repair mechanisms following CRISPR-Cas9 induced DNA cleavage?

The two DNA repair mechanisms are non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ directly ligates DNA ends without a template, potentially introducing indels, while HDR uses a homologous DNA template to guide error-free repair.

What are some potential applications of CRISPR technology?

CRISPR technology has applications in genetic screening, cancer immunotherapy, therapeutic management of AIDS, developing assays for detecting SARS-CoV-2, and improving plant traits such as disease resistance and crop yield.

What ethical concerns are associated with CRISPR genome editing?

Ethical concerns include the genome editing of human embryos and somatic cells, which pose potential risks and ethical dilemmas, especially regarding the inheritance of gene-linked diseases and the implications of modifying human genetics.

How does the CRISPR system in bacteria help in recognizing and neutralizing viral DNA?

In bacteria, CRISPR arrays are transcribed into crRNA, which forms complexes with tracrRNA and Cas9. When these complexes encounter complementary viral DNA sequences, Cas9 cleaves the DNA, preventing the virus from transcribing and replicating.

Outlines

00:00

🧬 Introduction to CRISPR-Cas9: A Revolutionary Genome Editing Tool

The first paragraph introduces the groundbreaking CRISPR-Cas9 system, a genome editing technology that has transformed molecular biology with its precision and specificity. Discovered in Escherichia coli in 1987 by Atsuo Nakata, CRISPR consists of palindromic DNA sequences that serve as an adaptive immune system for prokaryotes, protecting against viral DNA and mobile genetic elements. The paragraph delves into the historical context of CRISPR, its discovery, and its natural function in bacteria, setting the stage for understanding its biotechnological applications.

05:03

🛡️ Mechanism of CRISPR-Cas9: From Bacterial Defense to Programmable Genome Editor

This paragraph explains the molecular mechanism of CRISPR-Cas9. It describes the transcription of CRISPR arrays into CRISPR RNA (crRNA) and the involvement of the Cas9 nuclease protein, which cleaves DNA at specific sites. The paragraph details the formation of effector complexes with crRNA and tracrRNA, and how these complexes target and neutralize viral DNA through the recognition of a protospacer adjacent motif (PAM). The summary also highlights the transition of CRISPR from a natural bacterial defense to a programmable genome editing tool, pioneered by Jennifer Doudna and Emmanuelle Charpentier, and the development of single guide RNA (sgRNA) that simplifies the system for use in various organisms.

10:07

🧬 Applications and Implications of CRISPR-Cas9 in Modern Biology

The final paragraph discusses the wide-ranging applications of CRISPR-Cas9 technology. It touches on its use in genetic screening, cancer immunotherapy, and the treatment of AIDS. The paragraph also mentions the potential of CRISPR in detecting SARS-CoV-2 and the ethical considerations surrounding genome editing in human embryos and somatic cells. It concludes with the transformative impact of CRISPR on agriculture by enhancing disease resistance and crop yield in plants, and the anticipation of its role in solving major diseases and issues in molecular biology.

Keywords

💡CRISPR-Cas9

CRISPR-Cas9 is a revolutionary genome editing technology that allows for precise and site-specific alterations to the DNA of living organisms. It is derived from a naturally occurring defense mechanism in bacteria and has been adapted for use in various eukaryotic species. In the video, CRISPR-Cas9 is highlighted as the most promising biotechnology of the past decade due to its potential to manipulate genetic information with unprecedented control.

💡Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

CRISPR refers to the short, repeated sequences of DNA nucleotides found within the genome of prokaryotes. These sequences are palindromic, meaning they read the same forwards and backwards. The script explains that CRISPR was first identified in Escherichia coli and plays a key role in the adaptive immunity of prokaryotes, protecting them from viral DNA and other mobile genetic elements.

💡Cas9

Cas9, short for CRISPR-associated nuclease protein, is an enzyme that can cleave DNA at specific nucleotide linkages. It is a critical component of the CRISPR-Cas9 system, functioning like a pair of molecular scissors to cut DNA at precise locations. The script discusses Cas9's role in both the natural defense of bacteria and the biotechnological applications of genome editing.

💡Palindromic Repeats

Palindromic repeats are sequences of DNA that are the same when read from one end to the other on both strands of the DNA helix. In the context of the video, these repeats are part of the CRISPR structure and flank the spacers, which are derived from foreign genetic elements. They are essential for the function of the CRISPR-Cas9 system.

💡Spacers

Spacers are the unique sequences found between the palindromic repeats in the CRISPR array. They are derived from foreign DNA, such as that from bacteriophages, and serve as a 'memory' of past infections for bacteria. In the script, spacers are highlighted as integral to the CRISPR system's ability to recognize and defend against foreign genetic elements.

💡Adaptive Immunity

Adaptive immunity is the type of immunity that an organism develops after exposure to an antigen, such as from a pathogen or vaccination. The script explains that the CRISPR system in prokaryotes is a form of adaptive immunity, allowing them to remember and defend against foreign DNA from previous infections.

💡CRISPR RNA (crRNA)

crRNA, derived from the transcription of the CRISPR array, is a key component of the CRISPR-Cas9 system. It guides the Cas9 enzyme to the target DNA sequence for cleavage. The video describes the process of crRNA formation and its role in the specificity of the genome editing process.

💡Single Guide RNA (sgRNA)

sgRNA is a fusion of crRNA and tracrRNA, which can be synthesized in the lab for use in genome editing. It serves as a guide that pairs with the target DNA sequence, enabling the Cas9 enzyme to locate and cut the DNA at the desired site. The script discusses the creation of sgRNA as a major breakthrough in harnessing the CRISPR-Cas9 system for biotechnological purposes.

💡Protospacer Adjacent Motif (PAM)

PAM is a short sequence unique to the viral genome that is recognized by the CRISPR-Cas9 system before DNA cleavage occurs. The script explains that the presence of a PAM is necessary for the Cas9 enzyme to bind and cut the DNA, ensuring that the editing process is specific and targeted.

💡Homology-Directed Repair (HDR)

HDR is one of the two pathways used by cells to repair double-strand breaks in DNA. It uses a homologous DNA template to guide the repair process, reducing the likelihood of errors. The video mentions HDR in the context of genome editing, where it can be used to incorporate new DNA fragments at the site of cleavage.

💡Non-Homologous End Joining (NHEJ)

NHEJ is the other DNA repair pathway mentioned in the script, which repairs double-strand breaks without the need for a homologous template. This can result in insertions or deletions of DNA sequences, creating variations in the repaired DNA. NHEJ is highlighted as a common pathway in eukaryotic cells and is contrasted with HDR in terms of its precision and potential for error.

Highlights

CRISPR-Cas9 system is the most promising biotechnology technique of the past decade, revolutionizing molecular biology with its precise and site-specific gene editing capabilities.

CRISPR was first discovered in 1987 in Escherichia coli, as Clustered Regularly Interspaced Short Palindromic Repeats.

CRISPR sequences are palindromic, meaning they read the same forwards and backwards on complementary DNA strands.

CRISPR is a key component of prokaryotic adaptive immunity, protecting against viral DNA, bacteriophages, and plasmids.

CRISPR arrays are formed by integrating foreign DNA from previous infections between palindromic repeats, allowing bacteria to remember past infections.

CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) form a complex with Cas9 protein to target and cleave complementary viral DNA sequences.

Cas9 nuclease cleaves DNA at specific sites guided by crRNA, using a protospacer adjacent motif (PAM) for recognition.

Jennifer Doudna and Emmanuelle Charpentier proposed using CRISPR-Cas9 as a programmable genome editing toolkit for humans and animals in 2012.

Single guide RNA (sgRNA) was developed by fusing crRNA and tracrRNA, simplifying the system for laboratory use.

CRISPR-Cas9 can target any 20 base pair DNA sequence by designing a complementary sgRNA, enabling precise genome editing.

DNA repair after CRISPR-Cas9 cleavage can occur through homology-directed repair (HDR) or non-homologous end joining (NHEJ).

HDR uses a homologous DNA template for accurate repair, while NHEJ can introduce insertions or deletions (indels).

CRISPR has vast potential applications, including genetic screening, cancer immunotherapy, and detecting SARS-CoV-2.

CRISPR can modify immune T cells to enhance cancer recognition and killing when reintroduced into patients.

CRISPR shows promise in managing HIV/AIDS by targeting the proviral form of the virus that conventional therapies cannot.

Ethical concerns and potential risks exist with CRISPR, particularly in editing human embryos and germline cells.

CRISPR technology in plants can improve disease resistance, phenotypes, traits, and crop yields.

The future of CRISPR holds the potential to cure diseases and address major global health issues.

Transcripts

We’ve examined a handful of biotechnology concepts  in previous tutorials, but now it’s time to  

introduce what is undoubtedly the most promising  technique in biotechnology of the past decade. The  

CRISPR-Cas9 system represents genome editing  technology that has revolutionized molecular  

biology, due to its precise and site-specific gene  editing capabilities, which essentially allow for  

an unprecedented level of control in manipulating  the genetic information of a living organism.  

How does this work mechanistically, and what are  its applications? Let’s get a closer look now,  

starting with some historical context. In 1987, Atsuo Nakata and his team of  

researchers from the Osaka University in  Japan first reported the presence of Clustered  

Regularly Interspaced Short Palindromic Repeats,  abbreviated as CRISPR, in the Escherichia coli  

genome. These refer to short, repeated sequences  of DNA nucleotides found within the genome of  

prokaryotes. These sequences are the same  when read from 5' to 3' on one strand of DNA  

and from 5' to 3' on the complementary strand, and  are therefore described as palindromic repeats,  

just the way that we refer to words like racecar  or kayak as being palindromes, because they are  

the same whether read forwards or backwards. This  was further reported in both Gram-positive and  

Gram-negative bacteria, along with archaea,  leading to the obvious question regarding  

the relevance of CRISPR to these organisms,  which drove research for some time. Later on,  

in the mid 2000s, the functionality and importance  of CRISPR was first realized in prokaryotes. As it  

turns out, the CRISPR system is a key component  of their adaptive immunity, which protects  

these prokaryotes from attack by viral DNA,  bacteriophages, and plasmids. That’s right, it  

may seem incredible, but even unicellular bacteria  have a very basic immune system. Recall from our  

studies in the immunology series that adaptive  immunity refers to the immunity that an organism  

acquires after exposure to an antigen, either  from a pathogen or vaccination. Vaccination,  

for example, results in a form of adaptive  immunity in humans, since the body is exposed to  

antigens, and forms antibodies in response, which  contribute to the development of the immunity. 

The way this works for bacteria is as follows. The  unique sequences that are nestled in between the  

palindromic repeats, which are called spacers, are  bits of DNA that are foreign, and do not belong to  

the bacterium, but instead originate from mobile  genetic elements, or MGEs, such as bacteriophages,  

transposons, or plasmids that have  previously infected the prokaryote.  

This was revealed by sequencing the spacers found  in the CRISPR system, which led to the hypothesis  

that this could be a defense mechanism employed  by bacteria to recognize foreign DNA elements.  

During a viral infection, bacteria acquire  a small piece of the foreign viral DNA,  

and integrate it into the CRISPR locus to  generate CRISPR arrays. These consist of  

duplicate sequences, which are the palindromic  repeats belonging to the bacterial genome,  

flanked by variable sequences, or spacers, which  again are from the foreign genetic elements.  

In this way, bacteria retain a memory,  so to speak, of a past infection. 

So although it was initially revealed as a  genomic component of bacteria and archaea,  

CRISPR has inspired a method of genome editing  that can be applied to various eukaryotic  

species. But before we get there, we first have to  understand the function of CRISPR in prokaryotes,  

because understanding the mechanism of its natural  function will be necessary in order to understand  

the way it is exploited to achieve genome editing  capabilities in humans and other organisms. 

Let’s take a look at a particular Streptococcus  bacterium which is being attacked by a  

bacteriophage. Once the viral DNA is injected into  the cell, a section of it can be incorporated into  

the bacterial genome, and as we mentioned, it  will be inserted between the repeated palindromic  

sequences. This will now be called a spacer.  So here we can see three different spacers,  

potentially from three different viruses,  sandwiched in between the repeated palindromic  

sequences. Now we have what is called a  CRISPR array. This CRISPR array can undergo  

transcription, to form CRISPR RNA, abbreviated  as crRNA, although this longer strand is called  

pre-crRNA. Then the protein Cas9 gets involved.  Cas refers to CRISPR-associated nuclease protein,  

and as we know, nucleases are enzymes  that are capable of cleaving DNA  

at specific nucleotide linkages, kind of  like a pair of scissors. In particular, Cas9  

is one of the nucleases found in Streptococcus  pyogenes, which is one of the most extensively  

researched and characterized CRISPR-associated  nuclease proteins, so this is the one we will be  

looking at here inside this bacterium. Now along with Cas9, there are also  

molecules of tracrRNA. These have sections  that are complementary to and therefore  

can anneal to the palindromic repeats. So for  each spacer and palindromic repeat, we end up  

with a complex consisting of that segment of  pre-crRNA, a tracrRNA, and a Cas9 protein.  

Then another enzyme called ribonuclease three,  or RNase III, will cleave the strand in between  

these complexes, leaving us with individual crRNA  complexes which we can call effector complexes.  

With these effector complexes formed, the cell  is now ready to defend against the invader  

whose genome produced that crRNA. If this  complex encounters a section of viral DNA  

that has a sequence which is complementary to this  crRNA, the nuclease enzyme will coordinate, and  

if it recognizes a short sequence unique to the  viral genome called a protospacer adjacent motif,  

or PAM, then it will snip both strands of the  DNA, just a few base pairs upstream from the PAM.  

In doing so, it will neutralize the  virus, because its genome can no  

longer be transcribed properly to create more  viral particles, so infection is impossible. 

So that gives us a reasonable understanding of  how CRISPR is employed by prokaryotic organisms  

as a natural defense. Now it’s time to understand  how this phenomenon came to serve as the basis for  

biotechnological application. This begins in  2012, when Jennifer Doudna, a molecular biologist  

from the University of California, Berkeley  along with French microbiologist Emmanuelle  

Charpentier, were the first to propose  that the bacterial CRISPR-Cas9 system  

could be used as a programmable toolkit for  genome editing in humans and other animal species,  

and they eventually received the Nobel  prize in chemistry for their work, in 2020. 

So how can genome editing be achieved using this  method? The first thing we need to understand  

is that in bacteria, the crRNA and tracrRNA are  separate molecular entities. The first major  

breakthrough arrived when it was realized that  the roles of these molecules could be combined  

into a single molecule by fusing them together  with a linker to generate something called  

single guide RNA, or sgRNA, which  can be synthesized in the lab.  

If the sgRNA complexes with a Cas9 protein, this  two-component system will be able to cleave DNA  

just as the three-component system does in  bacteria. What this means was that it was then  

possible to determine any sequence of about 20  base pairs as a target for editing, and all that  

has to be done is to synthesize the appropriate  sgRNA with the complementary sequence, and insert  

that into a cell along with the Cas9 protein which  has been sourced from Streptococcus pyogenes.  

The complex will form, read the DNA until it  finds the appropriate sequence along with a  

PAM sequence, binding will occur, and DNA will  be cleaved at precisely the desired location.  

Cas9 has two domains, and each one  will snip one of the DNA strands. 

After the incision is made, the natural DNA  repair mechanism is enacted for the target DNA.  

The cleaved dsDNA can undergo repair via two  routes. Either by homology-directed repair,  

abbreviated as HDR, or by non-homologous end  joining, abbreviated as NHEJ. The NHEJ pathway  

repairs double-strand breaks in DNA by directly  ligating without the need for a homologous  

template, which means a DNA strand with similar  sequence that can act as a template. The NHEJ  

mechanism can also introduce insertion or deletion  of specific sequences at the joining ends,  

thus creating what are referred to as indels.  Indels are DNA strands with either an insertion  

or deletion of nucleotide sequences. Thus, NHEJ  produces DNA strands with non-uniformity in size. 

The other route of repair, the HDR pathway, is  commonly found in bacterial and archaeal cells,  

while the NHEJ pathway we just discussed is more  common in a eukaryotic domain. The HDR process,  

although more complex than NHEJ, uses a homologous  DNA template. The homologous DNA template has  

homology to the adjacent sequences surrounding the  site of cleavage to incorporate new DNA fragments.  

The template guides the repair process, and  lowers the possibility of errors. Since there is  

no insertion or deletion of nucleotide sequences,  the HDR pathway maintains uniformity in the size  

of the resulting dsDNA, unlike NHEJ. So that covers the mechanism of CRISPR  

genome editing technology. Now we move on  to the potential applications, which have  

only expanded ever since Doudna and Charpentier  suggested the possibility of using CRISPR for  

genome editing in humans and other animals. The  potential scope of application of CRISPR is vast,  

and includes its use as a genetic screen  to identify genes in different cells.  

One of the most prominent applications is in  cancer immunotherapy. In this practice, immune  

T cells, which are a type of white blood cell that  fights against a disease, are genetically modified  

using CRISPR technology. Specifically, these  T cells are extracted from the patient’s body  

and modified to make them more specialized in  recognizing cancer cells and killing them once  

they are reintroduced into the patient’s body. Similarly, CRISPR has also found its application  

in therapeutic management of acquired  immunodeficiency syndrome, or AIDS, which is  

caused by human immunodeficiency virus, also known  as HIV, as we covered in the microbiology series.  

Conventional anti-retroviral therapies are  capable of suppressing viral replication. But  

once the virus gets converted to its proviral  form, conventional therapies are ineffective  

in targeting the virus. The provirus resides  within the immune cells and continues to make  

copies of itself using the immune cell machinery,  and the immune cells fail to target the proviral  

latent reservoir which presents the risk  of viral rebound or relapse of the disease. 

Other than cancer and AIDS, CRISPR has also found  immense application in developing assays to detect  

SARS-CoV-2 infection, the cause  of the current global pandemic.  

Although genome editing of human embryos  and their implantation into a human womb,  

as well as genetic editing of somatic cells, have  wide ethical concerns and potential risks, CRISPR  

has the promise to cure various diseases and  prevent the inheritance of gene-linked diseases.  

Additionally, genome editing in  plants using CRISPR technology  

introduces the possibility of making  plants resistant to certain diseases,  

improving their phenotype or observable  characteristics, incorporating certain  

specific traits, improving crop yield, and so  forth. With so many invigorating possibilities  

for this exciting new technology, it will be  fascinating to see which of these major diseases  

and issues will be solved first, signaling  the dawn of a new era in molecular biology.