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 Table of Contents  
REVIEW ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 2  |  Page : 152-156

A new era in molecular biology clustered regularly interspaced short palindromic repeats/cas9 technology: A brief understanding


Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India

Date of Submission28-Dec-2020
Date of Decision16-Mar-2021
Date of Acceptance17-Mar-2021
Date of Web Publication14-May-2021

Correspondence Address:
Sejal Shah
Department of Microbiology, School of Science, R K University, Rajkot, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/aihb.aihb_162_20

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  Abstract 


Clustered regularly interspaced short palindromic repeats (CRISPR) are repeated patterns observed in bacterial DNA based on the natural defence mechanism of bacteria against any viral infection and plasmids. Targeted gene editing with the aid transcription activator-like effector nucleases and zinc-finger nucleases restricts its wide spared application due to convoluted protein structure designing. A CRISPR locus is organised of interspersed spacer and repeat sequences. Spacers are unique sequences originating from viral or plasmid DNA. CRISPR works in three phases (1) acquisition, (2) biogenesis and (3) targeting. Acquisition or adaptation involves the selection of foreign invading DNA (Protospacer). Biogenesis or expression and maturation integrate Protospacer into CRISPR loci transcribed and matured into crRNAs. During the targeting phase, crRNA forms a complex with cas9. There are three main classes of the CRISPR/cas9 system, i.e., type I, type II and type III, that have been discovered till date. All the data have been amalgamated through the following search engines such as PUBMED, Google Scholar and Medweb using keywords such as CRISPR/cas9 gene editing. The meta-analysis for the current study has been carried out by doing a systemic review starting from 2010 to 2017. CRISPR is currently the most adaptable and precise method employed for gene manipulation. Especially for the screening of mutant which increases in potency of T-cell cancer therapy, treatment for monogenic diseases, gene editing in embryos, zoonotic diseases such as malaria, eradication of HIV-1 genomes from T-cells can be possible. It can be used to understand how different genes influence disorders in the variety of animal systems. The natural bacterial protection mechanism is employed for gene editing due to expeditious scientific evolution and can be further availed for the treatment of various monogenic disorders. Embryo manipulation can be a divergent advancement in future, but due to social and ethical issues, this technique is to a halt.

Keywords: Cas9, clustered regularly interspaced short palindromic repeats, crRNA, gene editing, protospacer adjacent motif


How to cite this article:
Nalla Y, Shah S. A new era in molecular biology clustered regularly interspaced short palindromic repeats/cas9 technology: A brief understanding. Adv Hum Biol 2021;11:152-6

How to cite this URL:
Nalla Y, Shah S. A new era in molecular biology clustered regularly interspaced short palindromic repeats/cas9 technology: A brief understanding. Adv Hum Biol [serial online] 2021 [cited 2021 Sep 25];11:152-6. Available from: https://www.aihbonline.com/text.asp?2021/11/2/152/315952




  Introduction Top


The bacterial population is enormously huge in comparison to the human population and contains billions of DNA bases. Bacteriophages are viruses that infect and replicate within the bacterial cell either through the lytic cycle or lysogenic cycle.[1] These bacteriophages invade the bacterial cell, attach to the host cell, arrest the cell's protein machinery, replicate its DNA and start producing viral proteins. There are various strategies adapted by a microbe which helps it distinguish between self-DNA and foreign DNA and to survive from invasive elements.[2] These systems conserve the genetic element and succour the microbe in modifying self DNA with those changes which assist in survival. After a viral infection, bacteria coalesce new spacers which are extracted from the phage genome.[3] Clustered regularly interspaced short palindromic repeats (CRISPR) modifies the resistance of bacteria against phage and provides immunity [Table 1]. Gene-manipulating tools compromising with transcription activator-like effector nucleases (TALENs)[4] and zinc-finger nucleases[5] were the stepping stone in the field of gene manipulation which were based on the principle of DNA-protein recognition consisting with disadvantages such as protein synthesis, designing and validation. The term CRISPR which was coined in 2002[6] got in use for genome editing and enables genetic manipulation at specific target sit site in a single cell, tissues as well as whole organisms. Bacterial resistance, accomplished by CRISPR, also offers acquired immunity against different plasmids and viruses. CRISPR structure was inadvertently determined from Escherichia coli; similar structures were found in bacterial (~40%) and archaeal (~90%) genomes.[7] CRISPR is used to increase specificity which has RNA-guided (g-RNA) nucleases with the Cas9 protein system.[8] As genetic abnormalities can cause the certain disease like cancer,[9],[10],[11] there is a strong need of such tool to treat the disease.
Table 1: Three stages of adaptive immunity

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  Clustered Regularly Interspaced Short Palindromic Repeats System Top


CRISPR/Cas 9 works in three stages and acquire adaptive immunity” [Table 1].[12],[13],[14] The arrangement of CRISPR-Cas9 is built upon the principle of RNA-DNA hybridisation.[15] The RNA-guided genome editing is very useful for group II self-splicing introns to insert a particular sequence of interest at specific genomic loci.[16] Mutant generated from non-homologous end joining, caused either by insertions-deletions or through homology-directed repair, can be replaced with a targeted allele.[17] Likewise, other DNA repair pathways can also be included, The transactivating crRNA (tracrRNA) is responsible for the crRNA maturation through cas9 and ribonuclease II. The tracrRNA-crRNA complex initiates DNA cleavage and is together termed as sgRNA (single guide RNA).[18] The sgRNA (at 5' end) recognises the specific DNA sequence, whereas the 3' end guides the cas9 protein to bind to a specific DNA sequence.[19] TALENs and ZNFs associate with protein engineering for site-specific modification, whereas CRISPR necessitates only alteration in the guide RNA sequence. 'CRISPR interference' i.e., CRISPRa (activation) and CRISPRi (repression) use a catalytically sedentary Cas9, known as dCas9, which is fused with transcriptional activators and repressors.[15]

Clustered regularly interspaced short palindromic repeats loci

CRISPR loci comprise of cas (CRISPR associated) genes and disconnected direct repeats separated by intergenic spacer (non-coding DNA between genes) DNA segments.[20] DNA fragments are transcribed into RNA and then incorporated with cas proteins. The size of CRISPR repeats varies from 24 to 47 base pairs and repeats vary from 26 to 72 base pairs [Figure 1].[21] Fuction of various Cas genes has been listed in [Table 2].[32],[33],[34]
Table 2: ‘Function of various cas genes

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Figure 1: Clustered regularly interspaced short palindromic repeat locus: Cas gene cassettes encoding cas protein with leader/promoter sequence, followed by the number of total repeats (24–47 bp) and Spacers (26–72 bp).

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  Cas Proteins Top


There are many types of cas genes, but the most well-known is Cas 9 which was first observed in Streptococcus pyogenes (SpyCas9) and used as nickases, dual nickases and FokI fusion as alternatives.[22] CRISPR consists of six cas proteins, out of which cas1 and cas2 are united. Beyond these six genes, there are specific 'repeat-associated mysterious proteins' which pair with CRISPR repeat sequences.[23] These alternating spacers repeat sequences are conserved across all species. Cas proteins use guide RNA duplex sequence tracrRNA: CrRNA which makes complementary base pairing with the target DNA fragment causing site-specific double-stranded breaks at the desired site of DNA, thereby guiding the cas proteins to cut and paste the target DNA sequence.[24] Cas genes function as helicases, nucleases, polymerases and polynucleotide-binding proteins. With the help of Cas proteins, spacer DNA is identified, and cleavage of the invader DNA takes place.[25] The system attaches to the invader DNA, Cas9 unzips the genome and pairs it with the target RNA and these two molecules will cause cleavage.[26] Cas9 which was the first determined protein consists of two putative nuclease domains such as HNH and RuvC-like. HNH slices the complementary DNA strand to 20 nucleotides of crRNA and RuvC-like slices at the opposite of the complementary strand. Mutation in important domains such as RuvC-like or HNH of Cas9 produces several proteins with DNA (single-stranded) cleavage (nickase) activity.[27] Cas9 protein undergoes conformational rearrangement when it binds to guide RNA and further binding with dsDNA. There is another class of CRISPR effector proteins known as Cpf1 proteins and C2c1; there were a total of 16 Cpf1 enzymes discovered, out of which only two were reconcilable with human cells. Cpf1 has two advantages: (1) it is a single g-RNA molecule, small in size which can cause DNA break at multiple choices. (2) In contrast to Cas 9, it creates sticky ends, which makes Cpf1 more accordant in comparison to Cas9.[28],[29] Catalytically inactive Cas9 can be tagged with fluorescent proteins which can help in direct visualisation of genomic loci in live cells.[30] There are various types of bioinformatics tools which help in designing gRNA such as Cas9 Design, CRISPR MultiTargeter, CRISPR Primer Designer, GT-Scan, Off-Spotter, MIT Optimised CRISPR Design, sgRNA Designer, etc.[31]


  Protospacer Adjacent Motif Sequence Top


Endonuclease Cas9 distinguishes a conserved sequence of 2–4 base pairs, known as the protospacer adjacent motif (PAM). The invading DNA consists of this short PAM sequence pattern which is proximate to the crRNA. The cas system (type I and type II) is PAM dependent for their mechanism hence [Table 3], complementary base pairing with an invader target DNA and guide RNA end up with DNA unwinding and R-loop formation results in to blunt double-stranded DNA breaks.[6],[16],[35] Besides guide RNA–target DNA and PAM-based DNA recognition, conformational alternation of DNA cleavage disturbs Cas9 affinity [Figure 2].[36]
Table 3: ‘Stages of clustered regularly interspaced short palindromic repeats system’

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Figure 2: General mechanism of clustered regularly interspaced short palindromic repeat-Cas9 system and its three stages: (1) Viral DNA consists of a unique PAM sequence which is integrated into the CRISPR loci (2) Transcription takes place and crRNA is formed (3) crRNA binds with the cas protein and causes break down of viral genome. When the viral DNA enters the bacterial cell, the protospacer and unique PAM sequence help guide RNA and Cas9 to locate the sequence, followed by transcription of crRNA.

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Mechanism and types

To obtain nucleic acid recognition and cleavage, three types of CRISPR Cas mechanisms have 11 or more subtypes.[37] The type I and type III mechanisms involve complex Cas proteins, in contrast type II mechanism works with only single protein.[38]

There are mainly three types of mechanism followed for the gene-editing using CRISPR system [Figure 3].
Figure 3: Types of clustered regularly interspaced short palindromic repeats system: The crRNA is formed and three different types of clustered regularly interspaced short palindromic repeats mechanism take place. Type I and Type II are PAM dependent and Type III is PAM independent. In type II, two RNA sequences tracrRNA and crRNA form a complex known as guide RNA, which is the determining factor for target sequence cleavage.

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Type I-CASCADE complex is used for viral defences, which degrades the foreign DNA using a series of Cas proteins such as Cas6, Cas6e and Cas6f, whereas, Cas3 functions as the nuclease.[18] Type I system is the most frequently used and PAM-dependent mechanism where there is a formation of ribonucleoprotein complex with cascade which is made by mature crRNA.[42]

Types II Cas9 system: Type II system is PAM and TracrRNA dependent where tracrRNA base pairs with pre-crRNA transcript and makes a duplex.[43] Further, it binds to Cas9 forming a complex of tracrRNA: CrRNA: Cas9. Cleavage of DNA is undertaken by RNaseIII in Cas9 dependent manner.[44]

Type III Cmr-Csm complex: Pre-crRNA is cleaved by cas6. Type III system is further divided into two: III A system, in which Csm make a complex with mature cr RNA using double-stranded DNA while type III B system uses Cmr protein using RNA.[45]


  Challenges Top


For genome modification with the aid of the Cas9 enzyme, it is very important to select an optimising RNA guide and a unique target sequence.[46] Although there is a certain repairing mechanism, it has a high risk of mutations end up with a loss of function of the gene. CRISPR-Cas9 system interferes with a different mechanism, recruits other regions which cause off-target effects, and a large number of unexpected mutations occur.[47] The complexity of DNA decreases the septicity of CRISPR-Cas9. The International Bioethics Committee of UNESCO contemplates that the genome modification by CRISPR Cas9 should be strictly regulated and should only be implicated as a preventative measure to cure irremediable disease.[48] Until now, this technique has opened a new horizon to treat diseases like sickle cell anaemia, several types of cancer and cystic fibrosis. As cancer is a polygenic disease, the limitation is there of this tool to edit the genomic profile of the cancer patient.[49] Human clinical trials have been a great ethical issue worldwide as CRISPR may produce off target mutations resulting in cell death or transformation. Careful measures should be taken to prevent ecological damage and disequilibrium as well as to maintain human health.[50] Patenting has also been a major issue for scientists and companies for the trial of this technique on humans.[51]


  Conclusion and Further Prospective Top


Genome editing and manipulation can be done in somatic cells of mammals by introducing any DNA in the germ line of an organism.[52] New antimicrobials can be engineered which can target clinical genotypes or epidemiological isolates.Gene drive, which is a phenomenon of the introduction of a new genetic trait via a population run in a Non-Mendelian manner can be performed via CRISPR.[37] Hence, the disease can be controlled using disease-carrying genetic biomodels with gene drives.[53] It can be used for regulating the expression of the gene, gene knocking-screening in human cells by hindering the transcription-elongation process or inactivation of the gene by methylation of targeted DNA, multiplex genome engineering.[54] Alterations in animals and plants can be done to produce animal models, disease resistant, improve crop yield and quality or desired quality organisms.[55] Genetic mutations for different cancers can be repaired and overall landscape of patients can be engineered for better prognosis of the cancer patients; booming era for the genetic disorders.[56]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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