Name: crispr-cas9 and dna repair in cells
Uploaded: 2025-06-12T09:30:53.000Z
Duration: 1 min 28 s
Channel: JoVE

K12 Standards

Virginia  Standards of Learning

HIgh School

Biology

BIO.4

BIO.4.a

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system protects the bacteria from foreign genetic elements.In laboratories, the system is programmed using Cas-9 to edit genes in plants, animals, and humans.Cas9, an endonuclease from Streptococcus pyogenes, is delivered into the cells with a synthetic guide RNA- sgRNA.The sgRNA guides Cas9 to the Protospacer Adjacent Motif sequence, allowing it to bind and cut the DNA at the target site.For gene insertion, Cas9, guided by one sgRNA, cleaves the site, allowing homologous recombination to insert the new gene.For gene deletion, two sgRNAs direct Cas9 to cut both ends of the target region.The gene is excised, and the repair system joins the cut ends.CRISPR-Cas9 has been designed to inactivate HIV by excising its DNA from infected cells. Engineered systems like Csy4 target free HIV RNA, though these methods are still experimental.Besides targeting a single locus, CRISPR-Cas9 can edit multiple genes simultaneously&#8212;such as removing multiple retroviral copies.

crispr/cas9 genome editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing site-specific double-stranded breaks (DSBs) in DNA. In laboratory applications, CRISPR-Cas9 is reprogrammed to modify the genomes of diverse organisms.The system uses a synthetic single guide RNA (sgRNA) that directs Cas9 to precise genomic loci. The sgRNA contains a complementary sequence that guides Cas9 to a specific DNA target adjacent to a Protospacer Adjacent Motif (PAM) sequence, typically 5'-NGG-3' in the case of Cas9. Once binded, Cas9 induces a double-stranded break.The DSB facilitates homologous recombination for gene insertion when a donor DNA template is provided, leading to the precise integration of new genetic material. In gene deletion, two sgRNAs guide Cas9 in excising a defined DNA fragment by generating DSBs at both ends of the target region. The excised region is removed, and the cell's repair machinery rejoins the flanking sequences through non-homologous end joining (NHEJ), often resulting in a knockout.Beyond single-gene edits, CRISPR-Cas9 enables multiplexed genome editing, allowing simultaneous modification of multiple genes, which is advantageous in complex genetic studies and therapeutic applications. Notably, CRISPR-Cas9 has been employed to combat viral infections, such as HIV, by excising integrated viral DNA from host genomes. Other CRISPR-associated systems like Cys4 have also been developed to target and degrade free viral RNAs, such as HIV, broadening the scope of antiviral strategies.

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for ...

Education

JoVE Core

Microbiology

Unit

Viruses

KEY TERMS AND CONCEPTS

crispr-cas9 and dna repair in cells

CRISPR-Cas9 uses a bacterial defense system to change DNA in cells. The CRISPR-Cas system first evolved in prokaryotes to protect bacteria from invading genetic material such as viruses and plasmids. Scientists later adapted it into a genome-editing tool for plants, animals, and humans.

The key enzyme is Cas9, a DNA-cutting protein called an endonuclease from Streptococcus pyogenes. In the lab, a synthetic single guide RNA, or sgRNA, directs Cas9 to a chosen DNA site. The sgRNA matches the target sequence and works next to a Protospacer Adjacent Motif, or PAM, which is usually 5'-NGG-3' for Cas9.

After Cas9 binds, it makes a double-stranded break, or DSB, in the DNA. If scientists provide a donor DNA template, the cell can use homologous recombination to insert new genetic material at the cut site. This makes precise gene insertion possible.

CRISPR-Cas9 can also remove DNA. In gene deletion, two sgRNAs guide Cas9 to cut on both sides of a target region. The cell removes the excised piece and joins the remaining DNA by non-homologous end joining, or NHEJ. This repair often creates a knockout, which means the gene no longer works normally.

The system can edit more than one gene at the same time, which is called multiplexed genome editing. That makes it useful for complex genetic studies and possible therapies. CRISPR-Cas9 has also been used against HIV by cutting out integrated viral DNA from host genomes. Other CRISPR-associated systems, such as Cys4, can target and break free viral RNA, including HIV RNA, which expands antiviral strategies.

CRISPR-Cas9 and DNA Repair in Cells

introduction to virus

Virus Structure, Entry, and Replication

Viruses are biological entities that rely on host cells to make more copies of themselves. They do not have cells or their own metabolism, but they can show signs of life when they infect a host. Their basic structure includes a nucleic acid core, made of either DNA or RNA, surrounded by a protein coat called a capsid. Viruses are classified by the type of nucleic acid they contain and by their structure. Some viruses also have a lipid envelope that comes from the host cell membrane. Enveloped...

size and structure of viral genomes

How Viral Genomes Replicate

Viral genomes can be DNA or RNA, and their structure shapes how they replicate inside host cells. They may be linear or circular. They can also be single-stranded or double-stranded. These differences affect how a virus makes copies of itself and how it interacts with the host. RNA viruses usually have smaller genomes than DNA viruses. That smaller size is linked to high mutation rates and fast evolution. RNA virus polymerases often lack proofreading, so mistakes are more common. This helps...

viral replication: lytic cycle

T4 Phage Infection of E. coli

T4 phage infection of E. coli follows a lytic cycle that lets the virus make many copies fast. Bacteriophages, or phages, are viruses that infect bacteria, and T-even phages such as T4 are known for this well-studied process. The cycle ends with the destruction of the bacterial host. The first step is attachment to the E. coli cell surface. Tail fibers on the phage bind to receptor molecules on the bacterial outer membrane, which helps the virus find the correct host. After binding, the...

viral replication: lysogenic cycle

Prophage Integration in Bacterial Cells

The lysogenic cycle shows how a bacteriophage can stay inside a bacterial cell without killing it right away. Temperate phages, such as bacteriophage lambda (λ), use this strategy to persist in Escherichia coli while the host cell remains alive. After attachment to the bacterial surface, the phage injects its genetic material into the cytoplasm. The linear phage genome then circularizes, which helps protect it from host nucleases. Next, phage-encoded integrase carries out recombination that...

dna bacteriophages

How DNA Phages Replicate in Bacteria

DNA bacteriophages are viruses that infect bacteria and use bacterial cell machinery to make more copies of themselves. They carry either single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and each group uses a different replication strategy. Their life cycles also shape how they interact with hosts and how scientists use them in biotechnology and medicine. Some ssDNA phages keep their genomes very small and compact. The icosahedral phage ΦX174 shows this clearly because it uses...

viruses of archaea

Archaeal Virus Shapes and Extreme Survival

Archaeal viruses infect archaea that live in extreme places such as acidic hot springs and hydrothermal vents. These hosts often belong to the phyla Euryarchaeota and Crenarchaeota. The viruses help shape host evolution and gene transfer, which adds genetic diversity to microbial communities in harsh environments. Most archaeal viruses carry double-stranded DNA, or dsDNA, genomes. Some have single-stranded DNA, and some have RNA genomes. Their virions, or complete virus particles, show unusual...

viruses with rna genomes

RNA Virus Replication Strategies

RNA viruses use several replication strategies based on their genome type. They are grouped as positive-strand, negative-strand, or double-stranded RNA viruses. Some RNA viruses also use reverse transcription, which adds another layer to how they copy their genetic material. Positive-strand RNA viruses have genomes that can act like messenger RNA, or mRNA, as soon as they enter a cell. This lets the virus begin making proteins right away. Bacteriophage MS2 infects Escherichia coli by attaching...

the antiviral system of bacteria and archaea: crispr

the-antiviral-system-of-bacteria-and-archaea-crispr

How CRISPR Remembers Viral Infections

CRISPR is an adaptive immune system in bacteria and archaea that helps protect cells from viral infection. It works by identifying foreign genetic material, remembering it, and stopping the same invader later. In many cases, the main targets are bacteriophages, which are viruses that infect bacteria. The first defense step begins during an initial infection. Cas proteins, which are CRISPR-associated proteins, detect foreign DNA by looking for specific sequences called Protospacer Adjacent...

CRISPR-Cas9 uses a bacterial defense system to change DNA in cells. The CRISPR-Cas system first evolved in prokaryotes to protect bacteria from invading genetic material such as viruses and plasmids. Scientists later adapted it into a genome-editing tool for plants, animals, and humans. The key enzyme is Cas9, a DNA-cutting protein called an endonuclease from Streptococcus pyogenes. In the lab, a synthetic single guide RNA, or sgRNA, directs Cas9 to a chosen DNA site. The sgRNA matches the...

subviral agents

Viroids, Prions, and Satellite Viruses

Viroids, prions, and satellites are subviral agents that can cause infection even though they are not complete viruses. They resemble viruses in spread and disease, but each lacks one or more key viral parts, such as a capsid or the machinery needed for replication. Viroids are the simplest of these agents. They are circular, single-stranded RNA molecules with no protein coat. Viroids infect plants only and depend on host cell machinery to copy themselves. Host RNA polymerase replicates them...

key terms and concepts

Learn how CRISPR-Cas9 uses guide RNA, PAM sites, and cell repair pathways to insert, delete, or disable genes, including antiviral uses against HIV.

CRISPR-Cas9 and DNA Repair in Cells | JoVE K12

JoVE Core Microbiology provides an engaging introduction to the microbial world and its influence on health, industry, and the environment. This comprehensive collection covers key concepts such as microbial structure, metabolism, genetics, and ecology. It introduces essential laboratory techniques and microscopy methods through visually engaging animations. Corresponding scientist-in-action videos bridge classroom concepts with real-world research and applications.

microbiology

Introduction to Virus

Viruses are unique biological entities that blur the boundary between living and non-living systems. Although they lack cellular structure and metabolic ...

Size and Structure of Viral Genomes

Viral genomes exhibit remarkable diversity in size, structure, and composition, influencing their replication strategies and interactions with host cells. ...

Viral Replication: Lytic Cycle

Bacteriophages, or phages, are viruses that specifically infect bacteria. Among them, T-even bacteriophages, such as T4, exhibit a well-characterized ...

Viral Replication: Lysogenic Cycle

The lysogenic cycle is a crucial viral replication strategy that allows bacteriophages to persist within host cells without immediately destroying them. ...

DNA Bacteriophages

Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for ...

Viruses of Archaea

Archaeal viruses play a crucial role in the ecosystems of extremophilic archaea, particularly those belonging to the phyla Euryarchaeota and ...

Viruses with RNA Genomes

RNA viruses are categorized into positive-strand, negative-strand, or double-stranded groups based on their genomic structure and replication mechanisms. ...

The Antiviral System of Bacteria and Archaea: CRISPR

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects ...

Subviral Agents

Subviral agents are infectious entities that resemble viruses but lack one or more viral components, such as a capsid or essential replication machinery. ...

The student will investigate and understand that bacteria and viruses have an effect on living systems. Key ideas include  viruses depend on a host for metabolic processes;

Binary Fission

Binary fission is the primary mode of asexual reproduction in prokaryotes, such as bacteria. It results in the production of two genetically identical ...

Generation Time

Bacterial generation time, the period required for a bacterial population to double during its exponential growth phase, serves as a critical measure of ...

Bacterial Growth Curve

The bacterial growth curve is a fundamental concept in microbiology that describes the dynamics of bacterial population growth in a closed system with ...

microbial-growth-measurement-direct-methods

Microbial Growth Measurement: Direct Methods

Direct methods for measuring microbial populations in a culture are essential tools in microbiology, providing quantitative data for various applications. ...

microbial-growth-measurement-indirect-methods

Microbial Growth Measurement: Indirect Methods

Estimating microbial growth is essential for understanding population dynamics and environmental adaptations. Indirect methods provide valuable insights ...

Microbial Growth Media

Microbial growth media are essential tools in microbiology, providing the nutrients and conditions necessary to cultivate and study microorganisms. These ...

Need for Obtaining Pure Cultures

Pure cultures, defined as the growth of a single microorganism species isolated from mixed populations, are fundamental tools in microbiological research ...

techniques-for-isolation-of-pure-cultures

Techniques for Isolation of Pure Cultures

Microorganisms are routinely cultured in the laboratory using various techniques to isolate, grow, and quantify them for further study. These methods rely ...

Factors Influencing Microbial Growth: pH

Microorganisms are classified as acidophiles, neutrophiles, or alkaliphiles based on their pH growth preferences, reflecting their adaptations to specific ...

factors-influencing-microbial-growth-temperature

Factors Influencing Microbial Growth: Temperature

Microorganisms display remarkable adaptations, enabling them to thrive in diverse ecological niches across a wide range of temperatures. Temperature ...

factors-influencing-microbial-growth-osmolarity

Factors Influencing Microbial Growth: Osmolarity

Osmolarity is the measure of solute concentration in a solution. It plays a critical role in determining water availability for organisms. Water moves ...

Oxygen Requirements and Growth Patterns

Microorganisms exhibit diverse oxygen requirements and growth patterns driven by their metabolic strategies and environmental adaptations. Oxygen, while ...

Biofilms

Biofilms are complex communities of microorganisms encased in a self-produced extracellular polysaccharide matrix attached to surfaces. These microbial ...

The student will investigate and understand that bacteria and viruses have an effect on living systems. Key ideas include the modes of reproduction/replication can be compared

BIO.4.b

Prokaryotic vs. Eukaryotic Cells

Prokaryotic and eukaryotic cells represent two fundamental types of cellular organization, differing significantly in structure, complexity, and function. ...

Microbial Morphologies 

Bacterial and archaeal cells exhibit remarkable diversity in shape and structure, critical in their adaptability and functionality. Among bacteria, the ...

Bacterial Cell Wall 

The bacterial cell wall is an essential structural component that encases the plasma membrane, preserving cellular integrity, determining shape, and ...

Archaeal Cell Wall

Archaeal cell walls are structurally and compositionally distinct from their bacterial counterparts, lacking the characteristic peptidoglycan layer found ...

Peptidoglycan Synthesis

Structure of PeptidoglycanPeptidoglycan is a vital structural component of the bacterial cell wall, providing mechanical strength and shape to the cell. ...

Plasma Membrane in Bacteria and Archaea

The plasma membrane is an essential cellular structure responsible for maintaining cellular integrity and regulating the selective transport of molecules. ...

Outer Layers of the Cell Envelope

The outermost layers of prokaryotic cells play a critical role in their survival, virulence, and interaction with the environment. These layers, often ...

Flagella and Motility in Bacteria

Flagella are specialized, thread-like structures that extend from a bacteria's cell envelope. They play a crucial role in motility and chemotaxis. Their ...

Fimbriae, Pili, and Axial Filaments

Fimbriae and pili are specialized bacterial surface structures that play pivotal roles in adhesion, genetic exchange, and motility. Composed primarily of ...

Cell Inclusions

Prokaryotic cells possess a variety of inclusions that play crucial roles in nutrient storage, metabolic processes, and environmental adaptation. These ...

Endospores and Sporulation

Endospores are specialized, dormant cells primarily formed by Gram-positive bacteria, including Bacillus and Clostridium, enabling survival under extreme ...

Nucleoid

The nucleoid represents a structurally and functionally distinct region within prokaryotic cells, where the cell's DNA and associated proteins are housed. ...

Surface Appendages of Archaea

Archaeal surface appendages are highly specialized structures essential for environmental adaptation, encompassing roles in adhesion, biofilm formation, ...

The student will investigate and understand that bacteria and viruses have an effect on living systems. Key ideas include  the structures and functions can be compared

BIO.4.c

Types of Microorganisms

Microorganisms are a diverse group of microscopic entities broadly categorized into cellular and acellular types based on their structural organization. ...

environmental-applications-of-microorganisms

Environmental Applications of Microorganisms

Microorganisms play a pivotal role in maintaining ecosystem balance by recycling essential elements such as carbon, nitrogen, and phosphorus, as well as ...

community-dna-extraction-from-bacterial-colonies

Community DNA Extraction from Bacterial Colonies

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of analysis ...

detecting-environmental-microorganisms-with-polymerase-chain-reaction

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz
Polymerase chain reaction (PCR) is ...

rna-analysis-of-environmental-samples-using-rt-pcr

RNA Analysis of Environmental Samples Using RT-PCR

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Reverse ...

quantifying-environmental-microorganisms-and-viruses-using-qpcr

Quantifying Environmental Microorganisms and Viruses Using qPCR

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Quantitative polymerase ...

water-quality-analysis-via-indicator-organisms

Water Quality Analysis via Indicator Organisms

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona
Demonstrating Author: Luisa Ikner
Water quality analysis monitors ...

isolation-of-fecal-bacteria-from-water-samples-by-filtration

Isolation of Fecal Bacteria from Water Samples by Filtration

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
The quality of water destined ...

detection-of-bacteriophages-in-environmental-samples

Detection of Bacteriophages in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Alex Wassimi
Viruses are a unique group of ...

culturing-and-enumerating-bacteria-from-soil-samples

Culturing and Enumerating Bacteria from Soil Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Authors: Bradley Schmitz and Luisa Ikner
Surface ...

bacterial-growth-curve-analysis-and-its-environmental-applications

Bacterial Growth Curve Analysis and its Environmental Applications

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Bacteria are among the most ...

The student will investigate and understand that bacteria and viruses have an effect on living systems. Key ideas include  bacteria and viruses have a role in other organisms and the environment; and

BIO.4.d

History of Microbiology

Microbiology, a scientific field dedicated to the study of microorganisms, has undergone profound development since its inception in the 17th century. Its ...

Infection

When a pathogen enters the body and reproduces, it can cause an infection, damage body cells, and cause illness symptoms that eventually lead to disease. ...

Defense Mechanism Against Infection

Natural flora, body system defenses, and inflammation are natural barriers of the body against infectious agents regardless of previous exposure. Normal ...

The student will investigate and understand that bacteria and viruses have an effect on living systems. Key ideas include  the germ theory of infectious disease is supported by evidence.

BIO.4.e

CRISPR-Cas9 and DNA Repair in Cells

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