4.3 Advanced CRISPR tools and techniques
2 min read•july 25, 2024
CRISPR technology has evolved beyond its original form, with advanced systems like and Cas13 offering new capabilities. These tools expand CRISPR's reach, allowing for RNA targeting, enhanced precision, and improved delivery methods.
Enhancing CRISPR's and developing better delivery methods are crucial for its practical applications. , optimization, and diverse delivery methods like viral vectors and nanoparticles are pushing CRISPR's boundaries in research and therapy.
Advanced CRISPR Systems and Tools
Advanced CRISPR systems
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Frontiers | Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems View original
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Frontiers | Commentary: RNA editing with CRISPR-Cas13 View original
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Frontiers | Efficient Genome Editing in Populus Using CRISPR/Cas12a View original
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Frontiers | Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems View original
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Top images from around the web for Advanced CRISPR systems
Frontiers | Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems View original
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Frontiers | Commentary: RNA editing with CRISPR-Cas13 View original
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Frontiers | Efficient Genome Editing in Populus Using CRISPR/Cas12a View original
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Frontiers | Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems View original
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Frontiers | Commentary: RNA editing with CRISPR-Cas13 View original
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- Cas12a (Cpf1) functions as Class 2, type V CRISPR-Cas system recognizing T-rich PAM sequences generating staggered DNA cuts with 5' overhangs
- Cas13 targets RNA instead of DNA exhibiting collateral RNA cleavage activity with subtypes , , , and
- combines Cas9 nickase with reverse transcriptase enabling precise insertions, deletions, and base substitutions utilizing
Features of CRISPR tools
- Cas12a advantages include smaller size facilitating viral packaging and delivery, generating sticky ends enhancing insertion efficiency, processing its own crRNA enabling multiplex genome editing
- Cas13 benefits target RNA allowing transient gene regulation, useful for detecting and cleaving viral RNA, with potential applications in diagnostics and RNA editing (SHERLOCK)
- Prime editing achieves higher precision than traditional Cas9 editing reducing unwanted insertions or deletions capable of correcting up to 89% of known genetic variants (sickle cell anemia)
CRISPR Enhancements and Delivery Methods
Enhancing CRISPR specificity
- High-fidelity Cas9 variants:
- engineered to reduce non-specific DNA interactions
- contains mutations increasing specificity
- hyper-accurate with reduced
- naturally occur as inhibitors of CRISPR-Cas systems regulating Cas9 activity and improving control (AcrIIA4)
- Guide RNA optimization uses truncated guide RNAs to reduce off-target binding and chemical modifications to enhance stability and specificity
- Cas9 nickase strategy uses two Cas9 nickases to create double-strand breaks requiring two nearby target sites reducing off-target effects
CRISPR delivery methods
- Viral vectors:
- offers small packaging capacity but high efficiency with low immunogenicity and long-term expression
- provides larger packaging capacity integrating into the host genome
- Nanoparticles:
- encapsulate CRISPR components for efficient cellular uptake functionalized for tissue-specific targeting
- offer high cellular uptake efficiency conjugated with cell-penetrating peptides
- facilitate direct protein delivery of Cas9 and guide RNA reducing off-target effects by limiting exposure time (TAT peptide)
- introduces CRISPR components into cells effective for ex vivo applications and hard-to-transfect cells (T cells)
Key Terms to Review (33)
Adeno-Associated Virus (AAV): Adeno-Associated Virus (AAV) is a small, non-pathogenic virus that belongs to the parvovirus family and is commonly used as a vector in gene therapy. AAV can deliver genetic material into host cells efficiently and has a unique ability to integrate into the host genome, which makes it a valuable tool in advanced gene editing techniques, including CRISPR applications. Its safety profile and ability to transduce both dividing and non-dividing cells further enhance its utility in genetic research and therapeutic development.
Anti-crispr proteins: Anti-crispr proteins are proteins produced by certain bacteriophages that inhibit the CRISPR-Cas systems of their bacterial hosts. These proteins provide a defense mechanism for the phages, allowing them to evade the bacterial immune response and successfully infect their host cells. By understanding anti-crispr proteins, researchers can enhance CRISPR technology, making it more precise and controllable in genetic engineering applications.
Base editing: Base editing is a precise genome editing technique that enables the direct conversion of one DNA base into another without requiring double-strand breaks or donor DNA templates. This method leverages a modified CRISPR system to make targeted changes at specific genomic sites, allowing for more efficient and accurate gene editing compared to traditional methods. Base editing represents a significant advancement in the realm of CRISPR tools and techniques, offering the potential to correct point mutations that cause genetic diseases.
Bioethics: Bioethics is the study of the ethical issues arising from advances in biology and medicine, focusing on the moral implications of scientific practices and technologies. It examines how scientific developments impact individuals and society, addressing concerns such as safety, consent, equity, and environmental sustainability.
Biosecurity: Biosecurity refers to the measures and practices designed to prevent the introduction and spread of harmful biological agents, including pathogens and genetically modified organisms. It is essential in synthetic biology to ensure that engineered organisms do not pose risks to human health, agriculture, or the environment, highlighting the need for responsible innovation and oversight.
Cas12a: Cas12a, also known as Cpf1, is a CRISPR-associated endonuclease that plays a vital role in the CRISPR gene-editing system. It offers unique advantages over other Cas proteins, such as Cas9, including the ability to process its own guide RNA and create staggered DNA cuts, which can enhance precision in genetic modifications.
Cas13a: Cas13a is a type of CRISPR-associated protein that acts as an RNA-guided RNA endonuclease, primarily used for targeting and cleaving RNA molecules. It is part of the Class 2 CRISPR system and is distinct from Cas9, which targets DNA. Cas13a has gained attention in advanced gene editing and diagnostic applications due to its ability to selectively bind and degrade RNA, making it a powerful tool for manipulating gene expression and studying RNA biology.
Cas13b: Cas13b is a type of RNA-targeting CRISPR-associated protein that belongs to the Cas13 family, which is known for its unique ability to cleave RNA molecules. This protein has gained attention as an advanced tool in genetic engineering and synthetic biology, especially for its potential applications in gene editing and transcriptome manipulation.
Cas13c: Cas13c is a type of CRISPR-associated protein known for its RNA-targeting capabilities. It is part of the Cas13 family of proteins, which are distinct from the more commonly known Cas9 proteins, and play a significant role in advanced genetic engineering techniques. By targeting and cleaving RNA rather than DNA, Cas13c opens up new possibilities for manipulating gene expression and developing therapeutic strategies against RNA viruses.
Cas13d: Cas13d is a class 2 CRISPR-associated protein that functions as an RNA-guided endonuclease, specifically targeting RNA molecules for cleavage. This protein is derived from certain bacteria and has been adapted for various biotechnological applications, including gene regulation and targeted RNA editing. Its unique properties, such as smaller size compared to other Cas proteins and the ability to operate in eukaryotic cells, make it a powerful tool in advanced CRISPR techniques.
Cell-penetrating peptides (CPPs): Cell-penetrating peptides (CPPs) are short peptides that facilitate the transport of various molecular cargo across cell membranes. They possess unique properties that allow them to disrupt the lipid bilayer, enabling the uptake of therapeutic agents, nucleic acids, and proteins into cells, which is especially useful in gene editing and delivery applications.
CRISPR-Cas12: CRISPR-Cas12 is a groundbreaking genome editing technology that allows for precise modifications in the DNA of organisms. It operates as an advanced tool in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, utilizing a specific RNA-guided enzyme known as Cas12 that can create double-strand breaks in DNA. This system not only enhances the efficiency of genetic editing but also has expanded the applications of CRISPR technology in various fields such as medicine, agriculture, and biotechnology.
Crispr-cas13: CRISPR-Cas13 is a powerful RNA-targeting system that utilizes CRISPR technology for precise genome editing and regulation of RNA molecules. Unlike other CRISPR systems that target DNA, Cas13 focuses specifically on RNA, providing unique applications in gene expression modulation and viral defense. This technology has expanded the toolbox of genetic engineering by allowing researchers to selectively target RNA transcripts for degradation or modification.
CRISPR-Cas9: CRISPR-Cas9 is a revolutionary genome-editing technology that allows for precise modifications to DNA within living organisms. This tool has transformed genetic engineering by providing a simple and efficient method for targeting specific sequences in the genome, facilitating advancements in various fields including medicine, agriculture, and synthetic biology.
CRISPRa: CRISPRa, or CRISPR activation, is an advanced technique derived from the CRISPR-Cas9 system that enables targeted upregulation of gene expression. By utilizing a modified version of the Cas9 protein that lacks nuclease activity, CRISPRa can bind to specific DNA sequences and recruit transcriptional activators to enhance gene expression without altering the DNA sequence itself. This allows researchers to study gene function and regulation in a precise manner, making it a powerful tool in synthetic biology and metabolic engineering.
Crisproff: Crisproff is an advanced CRISPR-based technology that enhances the precision and efficiency of gene editing. It leverages improved guide RNA designs and modified Cas proteins to achieve higher accuracy in targeting specific genomic sequences, thus minimizing off-target effects and increasing the versatility of CRISPR applications in synthetic biology and metabolic engineering.
Electroporation: Electroporation is a technique used to introduce substances, like DNA or other macromolecules, into cells by applying an electric field to increase the permeability of the cell membrane. This method facilitates the transfer of genetic material, making it a vital tool in various applications, such as gene editing, therapeutic development, and cloning. By temporarily disrupting the lipid bilayer of the cell membrane, electroporation allows for more efficient uptake of foreign DNA, which is particularly useful in advanced genetic engineering strategies.
Espcas9: espcas9 is a modified form of the Cas9 protein used in CRISPR technology, specifically designed for enhanced gene editing efficiency and specificity. This variant includes specific mutations or alterations that improve its performance in targeting DNA sequences, reducing off-target effects, and increasing the precision of genetic modifications.
Gene editing: Gene editing is a biotechnological method that allows scientists to make precise changes to an organism's DNA, enhancing or altering specific traits. This powerful tool is revolutionizing fields like metabolic engineering, enabling the optimization of metabolic pathways and the creation of organisms with desirable characteristics for various applications.
Genome engineering: Genome engineering is a set of technologies that enable the modification of an organism's DNA to achieve desired traits or functionalities. This field combines molecular biology techniques with computational tools to edit, insert, or delete specific sequences within the genome, leading to applications in medicine, agriculture, and synthetic biology. One of the most significant advancements in genome engineering is the CRISPR-Cas9 system, which has transformed how scientists manipulate genetic material efficiently and accurately.
Germline editing: Germline editing is a form of genetic modification that alters the DNA in germ cells—sperm and eggs—so that the changes can be inherited by future generations. This technology opens up possibilities for eliminating genetic diseases, enhancing traits, and even raising ethical concerns regarding its long-term implications. It connects to various genome editing methods and is heavily associated with techniques like CRISPR-Cas9, which provide precise tools for these modifications.
Gold nanoparticles: Gold nanoparticles are tiny particles of gold that range in size from 1 to 100 nanometers. They have unique optical, electronic, and chemical properties due to their small size and high surface area, making them valuable in various applications, especially in biomedical fields like drug delivery and diagnostics. Their compatibility with biological systems allows for innovative uses in advanced techniques like CRISPR, where they can enhance gene editing efficiency and precision.
Guide RNA: Guide RNA (gRNA) is a crucial component in the CRISPR-Cas9 genome editing system that directs the Cas9 nuclease to a specific location in the DNA for targeted modifications. This RNA molecule consists of a short sequence complementary to the target DNA and a scaffold region that helps it bind to the Cas9 protein. The ability of gRNA to precisely locate and bind to specific DNA sequences is what makes CRISPR technology highly effective for genome editing.
High-fidelity cas9 variants: High-fidelity Cas9 variants are engineered versions of the Cas9 protein that have been optimized to enhance target specificity while reducing off-target effects in CRISPR gene-editing applications. These variants are crucial for precision editing, making them invaluable tools in genetic research and therapeutic development, as they minimize unintended changes to the genome.
Hypacas9: Hypacas9 is a modified version of the CRISPR-associated protein Cas9 that has been engineered for enhanced specificity and reduced off-target effects in genome editing applications. This advanced tool allows researchers to precisely target and edit specific DNA sequences with improved accuracy, making it a valuable asset in both basic research and therapeutic contexts.
Lentivirus: A lentivirus is a type of retrovirus characterized by its ability to integrate its genetic material into the host cell's genome, leading to long-term expression of the inserted genes. This feature makes lentiviruses particularly useful for gene delivery and therapeutic applications, including the development of advanced gene-editing tools that utilize CRISPR technology for precise modifications.
Lipid nanoparticles (LNPs): Lipid nanoparticles (LNPs) are tiny, spherical particles made of lipids that encapsulate therapeutic agents, such as mRNA or DNA, for efficient delivery into cells. They are crucial in advanced gene editing techniques, especially with CRISPR, as they enhance the stability and bioavailability of genetic materials, allowing for more effective and targeted therapies.
Off-target activity: Off-target activity refers to unintended modifications in the genome that occur when CRISPR-Cas9 or other genome-editing tools bind to and alter sequences that are similar, but not identical, to the intended target. This can lead to unexpected consequences in gene expression and function, raising concerns about the safety and specificity of these advanced editing techniques.
Prime editing: Prime editing is a revolutionary genome editing technology that enables precise and efficient modification of DNA sequences within living organisms. It combines the principles of CRISPR-Cas9 with a reverse transcriptase, allowing researchers to make targeted edits without causing double-strand breaks in the DNA. This advanced technique offers the potential to correct genetic mutations and perform gene therapies with higher accuracy and fewer off-target effects.
Prime editing guide RNA (pegRNA): Prime editing guide RNA (pegRNA) is a specially designed RNA molecule that directs the prime editing process to specific locations in the genome. It consists of a sequence complementary to the target DNA and an additional sequence that encodes the desired edit. This tool enhances the precision and versatility of CRISPR technology, allowing for accurate modifications without causing double-strand breaks, making it a significant advancement in genome editing techniques.
Regulatory compliance: Regulatory compliance refers to the adherence to laws, regulations, guidelines, and specifications relevant to an organization’s business processes. It ensures that the activities related to technology and product development meet established standards set by governing bodies, which is essential in areas like genetic engineering, industrial biotechnology, and protein production. By following these regulations, organizations can avoid legal repercussions and contribute to safety and ethical standards in scientific advancements.
Spcas9-hf1: spcas9-hf1 is a high-fidelity variant of the Cas9 protein used in CRISPR genome editing. This modified version has been engineered to reduce off-target effects while maintaining efficient on-target editing, making it a crucial tool in advanced genetic engineering applications. It connects with other CRISPR technologies by enhancing the precision of gene editing, which is vital for therapeutic applications and basic research.
Specificity: Specificity refers to the ability of a biological tool or system to accurately target and interact with a particular molecule or pathway while avoiding unintended interactions. This concept is crucial in various fields, including genetic engineering and biosensor development, where high precision is needed to ensure effective outcomes without cross-reactivity or false signals.