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.
All edits by Meredith Hanel
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 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)
- MAGESTIC (Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes) which can be used to study how sequence variants impact cell phenotypes on a large scale
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.
- Waxy corn enriched with amylopectin due to inactivation of endogenous waxy gene
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 which 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.
- Cas9 nickase, where Cas9 is modified to produce a single strand break and CRISPR-Cas9 nickases are delivered as pairs for more target specificity
- photoactivated Cas9 (paCas9) for optogenetic genome editing
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 which can be screened for a phenotype of interest. CRISPR libraries can knockout, activate or repress target genes. The following companies and non-profits sell or distribute CRISPR libraries.
- Twist Bioscience
- ThermoFisher Scientific
- Addgene (non-profit plasmid repository)
- anti-CRISPR protein AcrIIA4 and LOV photosensor for optogenetic control of CRISPR-Cas9
- Cas9 nickase
- Cas9 nickase, where Cas9 is modified to produce a single strand break and CRISPR-Cas9 nickases are delivered as pairs for more target specificity
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