CRISPR/Cas Tools
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins perform adaptive immune functions in prokaryotic organisms defending against foreign nucleic acids such as viruses. CRISPR/Cas tools have been adapted for use in genome editing and other DNA and RNA targeting applications.
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CRISPR/Cas Tools
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associatedCRISPR-associated (Cas) proteins perform adaptive immune functions in prokaryotic organisms defending against foreign nucleic acids such as viruses. CRISPR/Cas tools have been adapted for use in genome editing and other DNA and RNA targeting applications.
Class 1
The CRISPR-Cas systems belong to two classes, with multi-subunit effector complexes in Class 1 and single-protein effector modules in Class 2.
The effector complexes of type I and type III consist of a backbone of paralogous Repeat-Associated Mysterious Proteins (RAMPs) such as Cas7 and Cas5 with the RNA Recognition Motif (RRM) fold and additional large and small subunits.
Type I
Type III
Type IV
Class 2
The effector modules in classClass 2 are a single multi-domain protein and have a simpler and more uniform organization compared with classClass 1.
Type II
There are many variants of Cas9 from different prokaryotic species. Smaller sized versions of Cas9 are favorable for packaging and delivery into cells but there is a tradeoff in that smaller Cas9 proteins need a more complex PAM sequence which limits where they can target the genome. In parallel to research into gaining better understanding of and identification of new Cas proteins, there are efforts to re-engineer Cas9 proteins to reduce their size, increase fidelity and expand the target scope. CRISPR-Cas9 was the first CRISPR system used to edit human cells and is commonly used in genetic engineering and molecular biology applications.
Type IV
Type V
- CRISPR-Cas9—There are many variants of Cas9 from different prokaryotic species. Smaller-sized versions of Cas9 are favorable for packaging and delivery into cells, but there is a tradeoff in that smaller Cas9 proteins need a more complex PAM sequence, which limits where they can target the genome. In parallel to research into gaining better understanding of and identification of new Cas proteins, there are efforts to re-engineer Cas9 proteins to reduce their size, increase fidelity, and expand the target scope. CRISPR-Cas9 was the first CRISPR system used to edit human cells and is commonly used in genetic engineering and molecular biology applications.
Type VI
Type VI effectors all contain HEPN domains, which give them RNase activity, allowing them to target and degrade RNA.
CRISPR has revolutionalizedrevolutionized the genome editing field because it is simpler and more flexible to use than other genome editing tools such as meganucleases, ZFNs, and TALENs.
- Cas9 nickase, where Cas9 is modified to produce a single strand break, and CRISPR-Cas9 nickases are delivered as pairs for more target specificity
Genome editing with CRISPR-Cas9 has been demonstrated in model species such as Arabidopsis, rice, and tobacco as well as a few crop species. CRISPR-Cas12a genome editing has also been demonstrated to work in plants. The United States Department of AgricultureUnited States Department of Agriculture (USDA) stated that CRISPR-Cas9 edited crops will not be regulated as GMO.
- Drought tolerantDrought-tolerant soybean with edited Drb2a and Drb2b genes
CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are gene regulation tools that use a nuclease deactivatednuclease-deactivated version of Cas9 called dCas9. Gene expression is inhibited by dCas9 coexpressed with a guide RNA by interfering with RNA polymerase binding, transcriptional elongation, or transcription factor binding. When dCas9 is fused with the omega subunit of RNA polymerase or transcriptional activators, gene expression is enhanced.
The organization of genes within the genome and their location within chromatin architecture, such as chromatin loop structures, is important for gene regulation programs in development and disease.
- CLOuD9 is a dCas9 baseddCas9-based system, which selectively and reversibly establishes chromatin loops
- REPAIR RNA transcript editing system based on dCas13b fused to adensosineadenosine deaminase
- Cas12a basedCas12-based DETECTR system to detect viruses
- Cas12a basedCas12a-based HOMES system to detect viruses and small nucleotide polymorphism sites in human DNA related to health and personal characteristics
- Cas13a basedCas13a-based SHERLOCK to detect pathogenic viruses and bacteria
- Cas9Cas9- and Cas12 basedCas12-based systems by Caspr Biotech to detect viruses, bacteria, and genetic mutations
CAS-EXPAR (CRISPR/Cas9-triggered isothermal exponential amplification reaction) was developed to detect Listeria monocytogenes, a foodborne pathogen found in milk, milk products, eggs, poultry and meat which can cause invasive listeriosis and severe illness in young, elderly and immunocompromised individuals.
Genetic screening of mutant libraries is a way to search for genes involved in a desired pathway where mutant genes produce a certain phenotype. CRISPR libraries contain thousands of plasmids with multiple guide RNAs (gRNAs) for each target gene. Cells are treated with the library gRNA and Cas9, creating a population of mutant cells whichthat can be screened for a phenotype of interest. CRISPR libraries can knockout, activate, or repress target genes. The following companies and non-profitsnonprofits sell or distribute CRISPR libraries.
Anti-CRISPR proteins are a defense mechanism whichthat evolved in phages to escape destruction from CRISPR in their host prokaryotes. Anti-CRISPR proteins have applications as an “off switch” for CRISPR basedCRISPR-based genome editing or for reducing off-target cutting by CRISPR systems.
CRISPR/Cas systems are used to target sites of interest within the cell and attach a label, such as fluorescence, to allow them to be visualized.
- CRISPR/MB (dCas9 baseddCas9-based)
- CLING (dCas9 baseddCas9-based)
- CRISPR/Cas9 insertion of MS2 casettecassette for RNA tracking. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2 genome, which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA.
Recombineering is a method of genetic engineering in bacterial genomes that involves homologous recombination. In contrast to CRISPR used for gene editing in eukaryotic cells, CRISPR assists the selection of successful recombineering events whichthat change the target sequence and negatively selects cells in which the desired homologous recombination has not occurred. Negative selection occcursoccurs by CRISPR-Cas9 induced double strandeddouble-stranded breaks, which are lethal in bacteria where non homologousnon-homologous end joining (NHEJ) is not very effective.
Based on the ability of CRISPR systems to sequentially acquire DNA sequences from viral infections, adding them to spacer sequences in a growing array in the CRISPR locus, molecular recorder tools are being developed whichthat can record information provided by researchers or from the cell interactions with the environment.
- DNA based storage. Cas1 and Cas2 have been engineered by researchers at the Wyss Institute and Harvard Medical School to record pixel information in DNA so that the information could be retrieved to reconstruct frames in a galloping horse movie.
- CRISPR-Cas RNA recording system. A tool to record transient events, where genes are active and transcribing RNA, and record them in DNA. In order to accomplish this researchers at ETH Zurich, Switzerland fused a Cas gene to reverse transcriptase, which copies RNA into DNA.
- CAMERA (CRISPR-mediated analog multi-event recording apparatus) was developed using Cas9 nucleases and Cas9-derived base editors. The base editors were chimeric proteins comprised of a DNA base modification enzyme, a catalytically impaired Cas9 nickase and a base excision repair inhibitor. Their system was used to record events in bacteria or mammalian cells such as exposure to antibiotics, nutrients, viruses, light and changes in the signaling molecule, Wnt.
- DNA-based storage. Cas1 and Cas2 have been engineered by researchers at the Wyss Institute and Harvard Medical School to record pixel information in DNA so that the information could be retrieved to reconstruct frames in a galloping horse movie.
- CRISPR-Cas RNA recording system. A tool to record transient events, where genes are active and transcribing RNA, and record them in DNA. To accomplish this, researchers at ETH Zurich, Switzerland, fused a Cas gene to reverse transcriptase, which copies RNA into DNA.
- CAMERA (CRISPR-mediated analog multi-event recording apparatus) was developed using Cas9 nucleases and Cas9-derived base editors. The base editors were chimeric proteins comprised of a DNA base modification enzyme, a catalytically impaired Cas9 nickase, and a base excision repair inhibitor. Their system was used to record events in bacteria or mammalian cells, such as exposure to antibiotics, nutrients, viruses, light, and changes in the signaling molecule, Wnt.
CRISPR/Cas systems are used to target sites of interest within the cell and attach a label such as fluorescence to allow them to be visualized.
Genome editing with CRISPR-Cas9 has been demonstrated in model species such as Arabidopsis, rice and tobacco as well as a few crop species. CRISPR-Cas12a genome editing has also been demonstrated to work in plants. The United States Department of Agriculture (USDA) stated that CRISPR-Cas9 edited crops will not be regulated as GMO.
The following crops have been improved using CRISPR-Cas9 technology:
- Non-browning white button mushroom where polyphenol oxidase is knocked out
- Waxy corn enriched with amylopectin due to inactivation of endogenous waxy gene
- Green bristlegrass with delayed flowering due to inactivation of the ID1 gene
- Camelina with increased oil content by Yield10 Bioscience
- Drought tolerant soybean with edited Drb2a and Drb2b genes
Genome editing with CRISPR-Cas9 has been demonstrated in model species such as Arabidopsis, rice and tobacco as well as a few crop species. CRISPR-Cas12a genome editing has also been demonstrated to work in plants. The United States Department of Agriculture (USDA) stated that CRISPR-Cas9 edited crops will not be regulated as GMO.
The following crops have been improved using CRISPR-Cas9 technology:
- Non-browning white button mushroom where polyphenol oxidase is knocked out
- Waxy corn enriched with amylopectin due to inactivation of endogenous waxy gene
- Green bristlegrass with delayed flowering due to inactivation of the ID1 gene
- Camelina with increased oil content by Yield10 Bioscience
- Drought tolerant soybean with edited Drb2a and Drb2b genes
- CAMERA (CRISPR-mediated analog multi-event recording apparatus) was developed using Cas9 nucleases and Cas9-derived base editors. The base editors were chimeric proteins comprised of a DNA base modification enzyme, a catalytically impaired Cas9 nickase and a base excision repair inhibitor. Their system was used to record events in bacteria or mammalian cells such as exposure to antibiotics, nutrients, viruses, light and changes in the signaling molecule, Wnt.
Nucleic acid testing is used to screen and diagnose diseases and conditions. For nucleic acid testing, CRISPR systems are adapted to target nucleic acid sequences that are markers for disease and act as biosensors detecting pathogenic bacteria and viruses.
RAA-CRISPR/Cas12a (recombinase aided amplification assisted CRISPR/Cas12a) was developed to detect E. coli 05157:H7 and was shown to be accurate and sensitive in beef samples spiked with the bacteria.
CAS-EXPAR (CRISPR/Cas9-triggered isothermal exponential amplification reaction) was developed to detect Listeria monocytogenes, a foodborne pathogen found in milk, milk products, eggs, poultry and meat which can cause invasive listeriosis and severe illness in young, elderly and immunocompromised individuals.
CRISPR/Cas13a (APC-Cas) is a system that combines CRISPR with an allosteric probe (AP) that targets whole bacteria (Salmonella enteritidis).
Nucleic acid testing is used to screen and diagnose diseases and conditions. For nucleic acid testing, CRISPR systems are adapted to target nucleic acid sequences that are markers for disease.
- CRISPR-Cas RNA recording system. A tool to record transient events, where genes are active and transcribing RNA, and record them in DNA. In order to accomplish this researchers at ETH Zurich, Switzerland fused a Cas gene to reverse transcriptase, which copies RNA into DNA.
- Cas9 and Cas12 based systems by Caspr Biotech to detect viruses, bacteria and genetic mutations
- Programmed chromosome fissionProgrammed chromosome fission and fusionfusion in E. coli with Cas9
RecombineeringRecombineering is a method of genetic engineering in bacterial genomes that involves homologous recombination. In contrast to CRISPR used for gene editing in eukaryotic cells, CRISPR assists the selection of successful recombineering events which change the target sequence and negatively selects cells in which the desired homologous recombination has not occurred. Negative selection occcurs by CRISPR-Cas9 induced double stranded breaks which are lethal in bacteria where non homologous end joining (NHEJ) is not very effective.
- Programmed chromosome fission and fusion in E. coli with Cas9
- CRISPR/Cas9-based homology-independent targeted insertion (HITI)
- CRISPR/Cas9 insertion of MS2 casette for RNA tracking. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2bacteriophage MS2 genome which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA.
- CRISPR/Cas9 insertion of MS2 casette for RNA tracking. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2 genome which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA.
CRISPR-Cas systems are being developedcombined with phage therapy to target and degrade DNA of pathogenic bacteria as a selective antimicrobial treatment
CRISPR-Cas3CRISPR-Cas issystems are being developed by Locus Biosciences to target and degrade the DNA of pathogenic bacteria in orderas toa killselective it.antimicrobial treatment
- Locus Biosciences (CRISPR-Cas3)
- Eligo Bioscience (CRISPR-Cas9)
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