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Cas 9 and CRISPR: Systems, Mechanisms and Applications

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Introduction:

Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of prokaryotic DNA containing short repetations of base sequences. CRISPR systems are type of adaptive immune system confined to many bacterias for protection from foreign plasmids, viruses and also nucleic acids. They also protect bacterias from horizontal transmission of various genetic element. In CRISPR system the palindromic repeats are followed by spacer DNA , the short segment which is present due to previous exposures of bacteria to viruses and plasmids. They are found in 90% of archae and around 40% in bacterial genomes that has been sequenced . This CRISPR system has been widely used in addition , deletion, disruption and in changing the sequences of target genes collectively called gene editing and also in regulation of gene for species throughout the evolutionary tree. Cas genes code for proteins that are related to CRISPR system. Organism’s genome can be given a desired cut at desired location by injecting Cas protein and RNA’s in the cell. CRISPR systems are known to be used in stimulating biased inheritance of particular genes with an intention to alter entire population.

Presence of palindromic sequences were first introduced in year 1987 for bacterium E.coli. Later in 2000 clustered repeats were indentified in many of the bacterias and archae and were termed as short regularly spaced repeats (SRSR).IN 2002, the repeats (SRSR) were renamed as CRISPR. In 2005, it was revealed that CRISPR spacer were derivations of several bacteriophages and plasmids.These observations concluded that CRISPR system had a role to play in adaptive type of immunity in bacterias. It was also said that many of the spacers served as templates for RNA molecules while for eukaryotes these system is termed as RNA interference. Horvath, Moineau and Barrancou in year 2007, revealed that resistance to phages attack to streptococcus thermophilus organism can be altered using spacer DNA.

Cas 9 protein is known to induce DNA cleavage which leads to mutagenesis and is a result of error-prone non-homologus end joining (NHEJ). The DNA editing systems for Cas9 will help in revealing many virology related problems .Screening for human genes that are known to affect replication of pathogenic human viruses , derivations of clonal human cell lines which show absence of individual gene products are major applications.

The first technology that was developed and had a potential to be useful in the modification of large viral genomes was Zinc finger endonucleases. Zinc fingers are nearly 30 amino acids long domains that are potentially known to specifically recognise many but not all the 3 base pair long DNA stretches. Zinc finger endonuclease (ZFN) are fused to non-specific DNA endonuclease and thus resulting in DNA cleavage which would otherwise not be possible as ZFN themselves are incapable of cleaving the bound DNA.

Transcription activator like endonuclease (TALENs) are development based on distinct modular DNA binding motif. Zinc finger are not completely assured to be specific for all 3 base pairs target sequences and hence development of TALENs . Target sequences are known to be adjacent and present on both the DNA strands for greater degree of specific cleavage.

Diversity, ecology and evolution of CRIPR- Cas system :

The length and sequence of repeats and also the spacers are known to remain conserved with a locus of of CRISPR, but are likely to be variable between CRIPR in the same or different genome. Repeat sequences are found to be 21 bp to 48 bp and spacer are found to be 26 bp and 72 bp. There may be few to several hundreds of spacers found within a CRISPR locus. Single or multiple CRISPR loci are found in genomes , also at times in some species these loci becomes significant and major part of genomes.

CRISPRs in case of Methanocaldococcus species FS406-22 and Sulfolobus tokodaii strain 7 make up to 1% of total genome. Some CRISPR loci rely on transcoded factors in absence of adjacent cas genes. Conserved sequences called leader, are located upstream of CRISPR with respect to direction of transcription and are a added feature of some loci. Leader sequences was obsered for the first time in methanocaldococcus jannaschii, archaeoglobus fulgides and methanothermobacter thermoutotrophicus species but has subsequently confirmed in many other species.

Cas proteins are highly diverse group of proteins . proteins namely cas 1 and cas2 are involed in adaptation and are found virually universal in CRISPR-cas systemscas proteins are difficut in classification as they are present in wide variety , with presence of mutiple CRISPR loci. Different types of CRISPR- cas syatems are known to exist in organism . widely accepted classification of system are type I, II, III .New type IV system is proposed which show presence of several casade genes but no CRISPR cas 1 and cas 2. Type IV casade complex would be aided by protein DNA interaction but not by crRNA and is found to participate in innate immunity which is pre set to attack certain sequences.

Type I is recognised by protein cas 3 in which both helicase and DNase domains degrades the target. This type I system is further divided into six sub groups like type I A to type I F . These sub groups have variable number of cas genes. Other than cas i , cas 2 and cas 3 all type I systems are known to encode a cascade-like complex. Cascade binds to ctRNA and hepls in recognising target. In some cases , this cascade are known to enchance space acquistion. Cas 3 protein is the part of cascade complex in type IA system.

Type II CRISPR Cas system encodes for cas 1 and cas 2, cas 9 is labelled as signature protein, at times fourth protein namely Csn 2 or Cas 4. Adaptation, crRNA processing, clevage of target DNA site are all assisted by cas 9 protein followed by assistace of crRNA and additional RNA called tracr RNA .Type II systems have been divided into subtype IIA and IIB and recently into IIC . Type II A and type II C shows presence of csn 2 and cas 4 gene which aids in adaptation. Type II C lacks the presence of fouth gene.

Type III CRISPR-Cas system has cas 10 as its signature protein but the function of ot is unknown . Type I and type II are known to have involment in DNA targetting, and on other hand type III is known to target both DNA and/or RNA.

Type II system has been confined to only bacterial species while type I and type III are found in both bacterial and archael species.

Number of prokaryotic genomes are studied for presence of CRIPR and are used as a feature for argument on its importance in defence mechanism.It’s high prevalance is an effect of its mobile nature which is transferred horizontally and frequently.

Type IV system is similar to an ancestral innate immunity that gained adaptive ability by its accosiation with transposon-like element containing cas 1 and cas 2.

CRISPR mechanism:

Three types of CRISPR mechanisms have been identified so far. Amongst the three, type II is extensively studied. In this case , DNA from foreign organism like viruses or plasmids is cut into small fragments followed by incorporation into CRISPR locus admist a series of around 20bps. First the loci are transcribed and further the transcripts are processed to obtained small RNA labelled CRISPR RNA abbreviated as crRNA. These CRISPR RNAs are known to aid effector endonucleases that are the targets for invading DNA based on sequence complementarity.

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In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence.

Type 2 CRISPR system are very unique in comparison to other systems. In these system cas9 is a major participant in processing of crRNAs resulting in cleavage of intended DNA. Cas9’s function in both of the above mentioned steps relies on presence of two nuclease domains. A ruvC like nuclease domain which is present at amino terminus and HNH like nuclease domain that is present in the middle region of protein. Cas9 complexed with crRNA and separate trans activating crRNA, which is complimentary to crRNA will result in site specific DNA recognization and cleavage. crRNA maturation from a primary transcript which is known to encode multiple pre-crRNA is depended on tranc rRNA. Availability of RNAase III and cas9 is also the requisite.

During the destruction of target DNA presence of both HNH and RuvC like nuclease domains are responsible for cutting both DNA strands which results in generating double stranded breaks. These breaks are observed by 20 nucleotide target sequence which is within an crRNA transcript. RuvC domain cleaves the non complimentary strand and on the other hand HNH is known to cleave the complimentary strand.

Cas 9’s activity of being double stranded endonuclease is also depended on a short conserved sequence and it is known as protospacer associated motif and is abbreviated as PAM.

Cas 9 and CRISPR as a new tool in molecular biology:

Type II CRISPR nuclease is simple and is amenable for genome editing. Components of this system include cas9 , cr RNA, and trRNA.Scientist’s have synthesized a simplified two- component system by adding trRNA and crRNA into a single synthetic guide RNA (sgRNA) with reference model, typeII CRISPR system . Variants of cas 9 nuclease have been accepted in various protocols of genome editing . Wild type Cas 9 results in activating double strand break(DSB) repair machinary , which is known for site specific DNA clevage. Cell’s non homologus end joining (NHEJ) pathway repairs the DSB and results in deletions and/or insertions which disrupts the target locus. If we are able to supply donor template with homology of targeted sequence , the DSB is known to get repaired by homology directed repair mechanism. This allows replacement mutations to take place.

Cong and colleaques modelled the cas 9 system which showed increased precision by developing a mutant form labelled as Cas9D10A.this system cleaves only one strand of DNA and also NHEJ is not activated. When the system is provided with homologus repair template, DNA repairs are repaired via the HDR mechanism. In this case also indel mitations are reduced. Cas9D10A system is more applicative as it is target specific. This happens when loci are targeted by paired cas9 complexes which are intended to generate adjacent nicks in DNA.

Nuclease- deficient Cas9, dCas9 is a third type of variant. In D10A, RuvC domain and H840A in HNH domain inactivates the clevage activity thus preventing the DNA binding.This variant has application in sequence-specific target in any region of a genome without cleavage. By fusing various effector domains, dCas9 is known as either gene silencing or activation tool. It is also used as visualization tool.

Chen and colleaques fused dCas9 to Enchanced Green Fluroscent Protein (EGFP) for visualization of repetative DNA sequence by taking help of sgRNA.

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  1. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous endjoining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homologydirected repair pathway.
  2. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
  3. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Efficient programmable gene silencing by cascade:

Protocols that will allow change in expression of genes will enchance the tools for research in field of biology and medicine. Conventional methods confine change in individual particular gene , it’s regulatory element and even in mRNA’s translation rate. It is demonstrated that CRISPR-associated DNA- binding cascade complex can be put in use for efficient, long lasting and programmed gene silencing protocols. Trancription of down stream gene can be inhibited by targeting cascade to promoter sequences resulting in reduced gene expression.crRNA component helps in promoting specific cascade binding which helps in targeting particular stretch of DNA of interest. Gene silencing can be achieved by targeting cascade to the ORF sequence of gene, but at lower effiency. System is expected to silence targets with plasmids, chromosomes and also several genes as it is found active in different bacterial species and strains.

Gene expression should readily modulate to contantly changing environment. This rapid modulation is a key feature of any bacterial adaptive systems . Gene expression is regulated at two levels like trancriptional and post transcriptional in multilayer fashion. Activators and repressors alter the transcription initiation, by regulating themselves . mRNA once fored are subjected to variety of post trancriptional mechanisms. Discovery of riboswitches and riboregulators leads to conclusion that RNA plays a key role in regulation of gene expression. Regulation of RNA is mostly at post-trancriptional level and as such there are no known examples of RNA mediated control of promoter activity.

Genetic engginerring has helped to explore regulatory mechanisms for advancement in both basic and applied sciences. Enggineering sequence specificity of proteins is complex and time consuming task which makes it less applicable for simultaneous targeting in multiple genes. Programmed, simple and generic way of controlling transcription is desirable tool for cell and organism analysis.

CRISPR system becomes promising and efficient alternative for gene regulation. RNA guided DNA binding component is the major player for proving protection against foreign elements. CRISPR-Cas system has main two components (a) CRSPR locus (b) CRISPR associated genes. Expression of both the component will lead to defence mechanism.

CRISPR is transcribed leading to RNA processing by cas proteins into CRISPR RNA.

Crispr – Cas involment in processes other than immunity :

There are many evidences regarding CRISPR-Cas system’s involment in protection against genetic material but it has its involment in processes like regulation of virulence, genome evolution and also DNA repair. Their participation in above mentioned proccess are slowly becoming evident. In most cases, alternative functions for above processes are not well studied and further investigations are progressing for clarity of functions of many proceses.

It has been experimentally confirmed that cas 1 protein in E.coli aids in processing of single stranded and branched DNA species, replication forks and 5’ flaps. Interaction of cas 1 protein with recB, recC and ruvB has been confirmed suggesting its participation in DNA repair mechanism.CRIPR Cas gets triggered on accumulation of misfolded proteins in the membranes of E.coli, suggesting its active participation in “handling accumulation of defective proteins” pathway.

In Listeria monocytogenes, there is CRIPR without cas genes and hepls in targetting host chromosome and crRNA’S are known for increasing the level of target RNA by stabilizing it. It is also evident that in Framcisella novicida, repression of endogenous lipoprotein gene is mediated by type II CRISPR-cas system .Repression confines virulence of F. Novicida in mice as lipoprotein is known for triggering an innate type of response in mouse. Cas 9, trancrRNA and sca RNA works together for achieving repression. They bind to a region spanning the start codon of target’s gene. Increased virulence was observed when cas 9 protein was expressed in camplobacter jejuni lacking CRISPR loci . it has also been noted that cas 2 protein in legionella pneumophila is responsible for infection of amoebae in CRISPR- independent manner.CRISPR is also involved in inhibiting formation of biofilm in p. aeriginosa.

CRISPR applications:

In daiy industry, Daniso is marketing starting cultures, which on use will give improved cheese production and other application . these cultures contain bacterias that have CRISPRs with improved resistance resistance to various bacteriphages which would otherwise interfere with production and cause problems. It was the first company to realise the role of CRISPR-Cas system in immune mechanisms.

Development of type II systems is more applicable as a genetic tool for eukaryotic cells. Combination of crRNA and trancRNA to give sgRNA and this was the major outbreak of research related to CRISPR system. Cas 3 protein known to degrade the target sequence overtime, while cas9 gives a single double stranded break in DNA, these evidences aided in further researches regarding gentic editing tool.

CRISPR-Cas systems are soon becoming targets for plasmids which show presence of antimicrobial resistance markers. This also assures its application in obtaining antibiotic resistance strains. CRISPR’s are also developed with an potential application in development of strains refactory to uptake plasmids that carry undesirable genes. This also may be extended to develop starins so as to prevent uptake and dissemination of undesirable genetic elements such as prophages, anitibiotic resistane markers and photogenetic islands.

Requirements :

Desktop or laptop with either windows or mac operating system, internet access.

Softwares and databases: NCBI ,Softberry, Ecotools, Ecocyc

There are various steps involved in finding out the PAM and seed sequences .

Procedure:

The complete genome of E.coli MG1655 was downloaded from ecotools.

The repair and recombination genes were identified using Ecotools and NCBI databses.

Genes along with their promoter region was downloaded (ORF + 200 bp upstream of ATG corresponding to promoter)

The PAM sequences were highlighted using different colors.

The Eco cyc and softberrysoftwares were used to identiy the -35 and -10 regions.

Frequency of protospacer adjacent motif was determined in complete sequence and promoter and also their respective complements.

Identification of protospacer (32 base pairs upstream )were identified and documented.


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