CRISPR-Cas | Online Biotech Notes
Online Biotech Notes
CRISPR Cas-9
- Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 is a gene-editing technology causing a major upheaval in biomedical research. CRISPR-Cas9 enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence.
- CRISPR/Cas9 involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9)—an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome.
- It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.
HISTORY
➤1987-Osaka University researcher Yoshizumi Ishino and his colleagues found CRISPR sequences in Escherichia coli, but did not characterize their function.➤2000-CRISPR sequences are found to be common in other microbes byFrancisco Mojica, a scientist at the University of Alicante in Spain.
➤2002-Coined CRISPR name, defined signature Cas genes.
➤2007-First experimental evidence for CRISPR adaptive immunity was provided by a team of scientists led by Philippe Horvath.
➤2010-Study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus.
➤2013-First demonstration of Cas9 genome engineering in eukaryotic cell by Zhang lab.
➤2015-The nuclease Cas12a (formerly known as Cpf1) was characterized in the CRISPR/Cpf1 system of the bacterium Francisella novicida.
CRISPR-Cas Systems
• These are the part of the bacterial immune system which detects and recognize the foreign DNA and cleaves it.• The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci
• Cas (CRISPR-associated) proteins can target and cleave invading DNA in a sequence-specific manner.
• A CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes.
Spacer: The direct repeats in a CRISPR locus are separated by short stretches of non-repetitive DNA called spacers that are typically derived from invading plasmid or phage DNA
Protospacers: The nucleotide sequence of the spacer must be similar to the region in the phage genome called protospacer in order to recognize and subsequently block phage replication.
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| A CRISPR Locus |
Components of CRISPR
1. Protospacer adjacent motif (PAM)2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
Different CRISPR-Cas system in Bacterial Adaptive Immunity
Class1-type I ( CRISPR-Cas3) and type III (CRISPR-Cas10)
➢uses several Cas proteins and the crRNA
Class 2-type II (CRISPR-Cas9) and type V (CRISPR-Cpf1)
➢employ a large single-component Cas-9 protein in conjunction with crRNa and tracerRNA.
Different Cas Proteins and Their Function
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| Different Cas Proteins and their Function |
Action of CRISPR in Bacteria
1) Adaptation-leads to insertion of new spacers in the CRISPR locus.
2) Expression-the system gets ready for action by expressing the Cas genes and transcribing the CRISPR into a long precursor CRISPR RNA (pre-crRNA). The pre-crRNA is subsequently processed into mature crRNA by Cas proteins and accessory factors.
3) Interference-target nucleic acid is recognized and destroyed by the combined action of crRna and Cas proteins complex
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| Action of CRISPR in Bacteria |
Mechanism of CRISPR Cas9
• This system has been obtained from the bacteria and archaebacteria. When a bacteriophage infects bacteria, then the bacteria produces guide RNA which is complementary to a specific sequence of DNA of the virus and also produces another sequence of RNA. This sequence of RNA binds to the specific DNA sequence and the Cas9 identifies this sequence and cuts that portion by unwinding the DNA spiral.
CRISPR-Cas9 primarily consists of two molecules that are responsible for mutating the DNA. These are:
1. A Nuclease enzyme called Cas 9 which is responsible for cutting a specific sequence of DNA.
2. A Guide RNA which is basically a 20 base pair long RNA sequence that binds to the that has to be cut. It is complementary to the target DNA sequence. It in fact, directs Cas 9 to cut that sequence.
3. After the desired sequence is removed from the genome of the organism, the cell tries to naturally repair the cut in DNA but there are possibilities of mutation. These days, desired DNA sequences are also added in the reaction mixtures so that they get incorporated in the reaction mixtures.
This system has been used for manipulation of animal and plant cells genomes because CRISPR Cas 9 has a wide domain of scope. It can be used for deleting a mutated region which might be disease causing and adding the desirable sequence of DNA into the genome.
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| Deletion Of Mutated Region |
KeyAttributes that makes CRISPR system the ideal genome engineering technology
• High potency (cleavage efficiency) and specificity
• Broad applicability to both in vivo and ex vivo applications
• Simple editing tools (guide RNA plus protein) allow unprecedented ability to scale and optimize at speed
• Potential one-time curative treatment
• Ability to address any site in the genome or foreign genomes
• Ability to target multiple DNA sites simultaneously
• Multifunctional programmability: delete, insert or repair genes
Applications
●Human health and medicine-CRISPR-Cas9 has a lot of potential as a tool to prevent, treat, or cure medical conditions or diseases like cancer, hepatitis B, high cholesterol, diabetes, malaria,sickle cell disease and duchenne muscular dystrophy. CRISPR-Cas9 holds promise in combating antibiotic resistant pathogens.●Biomedical and clinical Research- Many of the proposed applications involve editing the genomes of somatic (non-reproductive) cells but there has been a lot of interest in and debate about the potential to edit germline (reproductive) cells because any changes made in germline cells will be passed on from generation to generation it has important ethical implications. The use of CRISPR-Cas9 and other gene editing technologies in somatic cells is uncontroversial, indeed they have already been used to treat human disease on a small number of exceptional and/or life-threatening cases.
●Agricultural Development-CRISPRCas9 permits the introduction or deletion of genetic sequences with much greater precision than traditional plant and livestock breeding techniques or earlier methods of genetic engineering.
Examples of Crops Modified with CRISPR Technology:
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| Examples of Crops Modified with CRISPR Technology |
●Industrial Biotechnology- First, CRISPR-Cas9 is broadening the number of microorganisms that could be used for industrial production. Second, CRISPR-Cas9 technology has been used to make industrially relevant strains resistant to viruses, to increase the production of chemicals used in biofuels, manufacturing, and to engineer probiotics.
●Ecosystem Management and Conservation-A CRISPR-based gene drive could be used by making an invasive species or an agricultural pest more susceptible to an herbicide or rodenticide, which would enable the species to be managed effectively by chemical control agents. It could also be used to bias the gender ratio of the invasive population towards males and therefore facilitate a decline in the population.
●Basic Research- CRISPR-Cas9 has made the development of animal models of disease less labor intensive, more cost-effective, and more precise. Before CRISPRCas9, creating a new mouse disease model took approximately a year and cost tens of thousands of dollars, but with the CRISPR technology a new mouse model can be created within a month and at a fraction of the previous cost. Beyond editing the genome (i.e., deleting and/or inserting genes), CRISPR-Cas9 is being used to regulate the expression of genes and the proteins they produce—providing additional insight into cellular systems and disease.
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