CRISPRs (clustered regularly interspaced short palindromic repeats) are DNAloci containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a virus.
CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryoticimmune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.
Since 2012, the CRISPR/Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates. By inserting a plasmid containing cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.
Bacteria may incorporate foreign DNA in other circumstances and even to scavenge damaged DNA from their environment.
Repeats were first described in 1987 for the bacterium Escherichia coli. In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encoding putative nuclease or helicase proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated genes).
Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats are shown as grey boxes and spacers are colored bars. While most CRISPR loci contain each of the three components, the arrangement is not always as shown.
In 2005, three independent researchers showed that CRISPR spacers showed homology to several phage DNA and extrachromosomal DNA such as plasmids. This was an indication that the CRISPR/cas system could have a role in adaptive immunity in bacteria. Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogously to eukaryotic cells that use a system called RNA interference.
In 2007 Barrangou, Horvath (food industry scientists at Danisco) and others showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.
Doudna and Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria deploy spacers in their immune defenses. They jointly studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to an invading phage by transcribing spacers and palindromic DNA into a long RNA molecule that the cell then uses tracrRNA and Cas9 to cut it into pieces called crRNAs.
Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home located its target DNA. Jinek combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Jinek et al proposed that such synthetic guide RNAs might be able to be used for gene editing.
CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012 It has since been used in a wide range of organisms including bakers yeast (S. cerevisiae), zebra fish, nematodes (C. elegans), plants, mice, and several other organisms.
Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.
Libraries of tens of thousands of guide RNAs are now available.
The first evidence that CRISPR can reverse disease symptoms in living animals was demonstrated in March 2014, when MIT researchers cured mice of a rare liver disorder.
In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to cut DNA at specific spots. Later, synthetic nucleases called TALENs provided an easier way to target specific DNA and were predicted to surpass zinc fingers. They both depend on making custom proteins for each DNA target, a more cumbersome procedure than guide RNAs. CRISPRs are more efficient and can target more genes than these earlier techniques.
CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.
CRISPR associated protein
crystal structure of a crispr-associated protein from thermus thermophilus
Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (~30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.
Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that CRISPR/Cas is a Lamarckian inheritance mechanism. Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.
Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.
The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012 provided a first step toward realization of proposals for CRISPR-derived biotechnology:
Artificial immunization against phage by introduction of engineered CRISPR loci in industrially important bacteria, including those used in food production and large-scale fermentation
Discrimination of bacterial strains by comparison of spacer sequences
Editas Medicine, a $43 million startup, aims to develop treatments that employ CRISPR/Cas to make edits to single base pairs and larger stretches of DNA. Inherited diseases such as cystic fibrosis, sickle-cell anemia and Huntington's disease are caused by single base pair mutations; CRISPR/Cas technology has the potential to correct these errors. The "corrected" gene remains in its normal location on its chromosome, which preserves the way the cell normally activate inhibits its expression.
After harvesting blood cell precursors called hematopoietic stem cells from a patient's bone marrow, CRISPR gene surgery would correct the defective gene. Then the gene-corrected stem cells would be returned to the patient's marrow, which would then produce healthy red blood cells. Replacing 70% of the sickle cells would produce a cure.
Before it can be used clinically, the company must be able to guarantee that only the targeted region will be affected and determine how to deliver the therapy to a patient’s cells.
Other pathologies potentially treatable by CRISPR include Huntington’s disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos.
Improved targeting is required before CRISPR can be used in medical applications. Current guide RNAs may target sequences that differ by multiple base pairs from the intended sequence.
CRISPR simplifies creation of mouse models and reduces the time required to a matter of weeks from months or longer. Knockdown of endogenous genes has been achieved by transfection with a plasmid that contains a CRISPR area with a spacer, which inhibits a target gene. Injecting mouse zygotes with Cas9 and two guide RNAs was able to disable two genes with 80% efficiency. So-called homology-directed repair involves using Cas9 to "nick" DNA, to introduce new gene parts to the zygote.
CRISPRs can add and delete base pairs at specifically targeted DNA loci. CRISPRs have been used to cut as many as five genes at once.
"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.
Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.
The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.
Use by phages
Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from bacterial chromosome upon phage infection and can inhibit phage replication. The mechanisms that induce PICI excision and how PICI inhibits phage replication are not well understood. One study showed that lytic ICP1 phage, which specifically targets Vibrio choleraeserogroup O1, has acquired a CRISPR/Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the 1-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows the phage to co-evolve with its host.
Automation and library support
Free software is available to design RNA to target any desired gene. The Addgene repository offers academics the DNA to make their own CRISPR system for $65. In 2013 Addgene distributed more than 10,000 CRISPR constructs. The facility has received CRISPR-enabling DNA sequences from 11 independent research teams.
A provisional US patent application on the use of the CRISPR system for editing genes and regulating gene expression was filed by the inventors on May 12, 2012. Subsequent applications were combined and on March 6, 2014 the resulting patent application was published by the USPTO. The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.
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