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CRISPR/Cas Tools

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.

Classification of CRISPR systems

Class 1

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 class 2 are a single multi-domain protein and have a simpler and more uniform organization compared with class 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

Type VI

Type VI effectors all contain HEPN domains which give them RNase activity allowing them to target and degrade RNA.

Gene/genome editing

CRISPR has revolutionalized the genome editing field because it is simpler and more flexible to use than other genome editing tools such as meganucleases, ZFNs and TALENs.

  • CRISPR-Cas9
  • CRISPR-Cas12a
  • Cas9 nickase, where Cas9 is modified to produce a single strand break and CRISPR-Cas9 nickases are delivered as pairs for more target specificity
  • Madzyme, Mad7 similar to Cas12a developed by Inscripta
  • photoactivated Cas9 (paCas9) for optogenetic genome editing
  • 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

Regulating gene expression

CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are gene regulation tools that use a nuclease 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.

Gene represssion

  • dCas9-KRAB

Gene activation

  • dCas9-VP64

Epigenome editing

Epigenetic CRISPR/Cas9 uses dCas9 fused with a protein that changes components of the epigenome, like chromatin structure and DNA methylation, resulting in upregulation or downregulation of gene expression.

  • dCas9-DNMT3A
  • dCas9-Ezh2
  • dCas9-DNMT3B
  • dCas9-TET1/2
  • dCas9-KRAB
  • dCas9-MQ1 (prokaryotic DNA methyltransferase)

Engineering chromatin loop structures

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 based system which selectively and reversibly establishes chromatin loops

RNA editing

  • REPAIR RNA transcript editing system based on dCas13b fused to adensosine deaminase

Diagnostics

  • Cas12a based DETECTR system to detect viruses
  • Cas12a based HOMES system to detect viruses and small nucleotide polymorphism sites in human DNA related to health and personal characteristics
  • Cas13a based SHERLOCK to detect pathogenic viruses and bacteria
  • CRISPR-on-a-chip for cancer diagnostics

CRISPR library screening

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.

Anti-CRISPR proteins

Anti-CRISPR proteins are a defense mechanism which evolved in phages to escape destruction from CRISPR in their host prokaryotes. Anti-CRISPR proteins have applications as an “off switch” for CRISPR based genome editing or for reducing off-target cutting by CRISPR systems.

  • anti-CRISPR protein AcrIIA4 and LOV photosensor for optogenetic control of CRISPR-Cas9

Live cell imaging

  • dCas9-GFP
  • CRISPR/MB (dCas9 based)
  • CLING (dCas9 based)
  • Cas13a fused with GFP for tracking RNA

CRISPR-assisted recombineering

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 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.

CRISPR antimicrobials

CRISPR-Cas3 is being developed by Locus Biosciences to target and degrade the DNA of pathogenic bacteria in order to kill it.

Agriculture

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

CRISPR-Cas molecular recording

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.

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