Open access peer-reviewed chapter - ONLINE FIRST

CRISPR Technology: Emerging Tools of Genome Editing and Protein Detection

Written By

Rita Lakkakul and Pradip Hirapure

Submitted: December 14th, 2021Reviewed: January 6th, 2022Published: April 1st, 2022

DOI: 10.5772/intechopen.102516

IntechOpen
Molecular CloningEdited by Sadik Dincer

From the Edited Volume

Molecular Cloning [Working Title]

Prof. Sadik Dincer, Dr. Hatice Aysun Merci̇mek Takci and Associate Prof. Melis Sümengen Özdenefe

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Abstract

CRISPR technology has seen rapid development in applications ranging from genomic and epigenetic changes to protein identification throughout the last decade. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the ability to edit, control the genomic nucleic acid and non-nucleic acid target such as detection of proteins. CRISPR/Cas systems are RNA-guided endonucleases exhibiting distinct cleavage activities deployed in the development of analytical techniques. Apart from genome editing technology, CRISPR/Cas has also been incorporated in amplified detection of proteins, transcriptional modulation, cancer biomarkers, and rapid detection of POC (point of care) diagnostics for various diseases such as Covid-19. Current protein detection methods incorporate sophisticated instrumentation and extensive sensing procedures with less reliable, quantitative, and sensitive detection of proteins. The precision and sensitivity brought in by CRISPR-dependent detection of proteins will ensure the elimination of current impediments. CRISPR-based amplification strategies have been used for accurate estimation of proteins including aptamer-based assay, femtomolar detection of proteins in living cells, immunoassays, and isothermal proximal assay for high throughput. The chapter will provide a comprehensive summary of key developments in emerging tools of genome editing and protein detection deploying CRISPR technology, and its future perspectives will be discussed.

Keywords

  • CRISPR/Cas
  • genome editing
  • protein detection
  • CRISPR technology
  • anti-CRISPR

1. Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein modules are found to be a part of adaptive antivirus defense systems in archaea and bacteria and mediate immunity by a three-stage process called adaption, processing of the primary transcript, and interference. These systems incorporate fragments of foreign DNA (known as spacers) into CRISPR cassettes, then transcribe the CRISPR arrays including the spacers, and process them to make a guide crRNA or the clustered regularly interspaced short palindromic repeats ribonucleic acid (CRISP RNA) which is employed to specifically target and cleave the genome of the cognate virus or plasmid. Earlier classic methods such as zinc finger motif, meganucleases, and transcription activator-like effector nucleases were deployed for genome editing but due to its prerequisite for different fusion proteins, the technology raised hurdles in its applicability. The characteristic feature of single guide RNA of CRISPR to regulate Cas protein to target specific gene sequence is highly advantageous to overcome the barriers posing from classic methods. Proteins cas1 and cas2 genes are found to be the core and active part of the information processing subsystems of the three distinct types of CRISPR/Cas systems [1]. Due to the current problems with the vast diversity and complexity of the architecture of CRISPR/Cas systems, the classification is still challenging. Based on the presence of three signature genes, the classification is as follows:

1.1 Type I CRISPR/Cas systems

Typical type I loci contain the signature cas3 gene, which codes for helicase and DNase activities within a single large protein. The detailed sequence and structural comparisons have led to the recognition of many of these proteins in the RAMP superfamily including Cas5 and Cas6 families. Type I systems are currently divided into six subtypes, I-A to I-F, each of which has its own signature gene and distinct features of operon organization [2, 3].

1.2 Type II CRISPR/Cas systems

These contain cas9 as a signature gene encoding for a multidomain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the maturation of the crRNA. These systems use cellular (not encoded within the CRISPR/Cas loci) RNase III and tracrRNA for the processing of pre-crRNA. Type II CRISPR-Cas systems are currently classified into three subtypes such as II-A, II-B, and II-C. Type II-A encompasses an additional signature gene csn2. Protein csn2 is found to be engaged in spacer integration. Type II-B systems belong to the Cas family of proteins with 5′-single-stranded DNA exonuclease activity. The recently proposed type II-C CRISPR-Cas systems possess only three protein-coding genes (cas1, cas2, and cas9) and are common in sequenced bacterial genomes (Figure 1) [2, 3].

Figure 1.

Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems. The CRISPR-Cas systems are composed of acasoperon (blue arrows) and a CRISPR array that comprises identical repeat sequences (black rectangles) that are interspersed by phage-derived spacers (colored rectangles). Upon phage infection, a sequence of the invading DNA (protospacer) is incorporated into the CRISPR array by the Cas1-Cas2 complex. The CRISPR array is then transcribed into a long precursor CRISPR RNA (pre-crRNA), which is further processed by Cas6 in type I and III systems (processing in type I-C CRISPR-Cas systems by Cas5d). In type II CRISPR-Cas systems, crRNA maturation requires tracrRNA, RNase III and Cas9, whereas in type V-A systems Cpf1 alone is sufficient for crRNA maturation. In the interference state of type I systems, Cascade is guided by crRNA to bind the foreign DNA in a sequence-specific manner and subsequently recruits Cas3 that degrades the displaced strand through its 3′–5′ exonucleolytic activity. Type III-A and type III-B CRISPR-Cas systems employ Csm and Cmr complexes, respectively, for cleavage of DNA (red triangles) and its transcripts (black triangles). A ribonucleoprotein complex consisting of Cas9 and a tracrRNA: crRNA duplex targets and cleaves invading DNA in type II CRISPR-Cas systems. The crRNA-guided effector protein Cpf1 is responsible for target degradation in type V systems. Red triangles represent the cleavage sites of the interference machinery (Courtesy: Ref. [4]).

1.3 Type III CRISPR/Cas systems

All type III systems possess the signature gene cas10 which encodes a multidomain protein containing a palm domain similar to that in cyclases and polymerases of the PolB family (Table 1) [2, 3].

ClassificationType IType IIType IIIType V
Signature proteinCas 3Cas 9Csm (III-A) or Cmr (III-B)Cas 12a
EffectorCascadeCrRNAsgRNACrRNA
Cleavage productSSBsDSBsSSBsDSBs

Table 1.

Different types of CRISPR/Cas based on signature protein, effector, and cleavage product [3].

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2. Molecular characterization of CRISPR-Cas 12 and Cas 13

Initially, CRISPR-Cas 9 was found to nick the DNA along with the guide RNA Cas 12a belonging to class II Type VA system, derived from Francisella novicidabacterium possesses enormous ability to cleave DNA at multiple targets. Cas 12 an RNA-guided DNAse, is a T-rich PAM sequence making it different from Cas 9. The positively charged central channel of a nuclease (NUC) domain determines the trans cleavage activity of the target strand after studies find that mutations in the catalytic site of the RuvC domain of Cas12a in the bacterium Acidaminococcussp. eliminate the same. CRISPR is classified into types I and II [5].

Type II is further divided into six types based on their structure and function. The Cas12a protein contains a RuvC endonuclease domain, which sequentially cleaves the non-targeting strand and the targeting strand to form DSBs (double-stranded base pairs). Compared to the CRISPR/Cas9 system, this system has several remarkable differences, including the signature protein, PAM sequence, and cleavage product [6]. CRISPR/Cas12a based sensing methods focus on fluorescence readout with reduced transduction efficiency as studies report a direct correlation between the catalysis systems with recognition elements (i.e., aptamers), thus greatly improving the working efficiency of the detection platform. Cas 13 consists of four subtypes and is involved in RNA interference activities. Off-target editing is critical to Cas 13 and requires significant attention in retrieving obstacles for protein analysis [7].

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3. Mechanism of amplification strategy for nucleic acid and protein detection

CRISPR/Cas systems generally play a role as RNA-guided endonucleases (crRNA). The crRNA guides cas proteins to specific DNA sequences whereby the hybridization leads to cas protein activation which later results in cleavage of DNA sequence [8]. Figure 2 shows the components of Cas9, Cas12a, Cas12f, and Cas13a.

Figure 2.

Basic components of CRISPR/Cas9, Cas12a, Cas12f, and Cas13a pink triangle indicates cis-cleavage site [9].

Cas9 is an endonuclease and the single-guide RNA (sgRNA) of CRISPR-Cas9 systems contains a hairpin-rich region that binds to Cas9 and a 20-nucleotide “spacer” region that binds with the complementary “protospacer” region in the target strand of a dsDNA duplex. Binding between the sgRNA and the DNA target brings Cas9 into close proximity to the target (Figure 2). The His-Asn-His (HNH) domain of Cas9 cleaves the strand that is complementary to sgRNA (target strand) and the RuvC domain of Cas9 cleaves the other strand of the dsDNA (non-target strand). Single-guide RNA (sgRNA) (Figure 3) [13].

Figure 3.

Combining functional nucleic acids and molecular translators with CRISPR/Cas technology for detection of non-nucleic acids such as proteins. Adapted from Ref. [10]. Copyright 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Adapted from Ref. [11]. Copyright 2020 American Chemical Society. Adapted from Ref. [12]. Copyright 2016 Springer Nature. (A) Two copies of an aptamer lock the activator, the target ssDNA complementary to crRNA. The activator is released when the aptamer binds to a small molecule (e.g., ATP), allowing it to hybridize with crRNA and activate CRISPR-Cas12a trans-activity. (B)The activation of CRISPR-Cas12a is prevented when the target molecule binds to its aptamer. CRISPR-Cas12a is activated by an unbound aptamer. (C) Metal ions serve as co-factor(s) for an RNA-cleaving DNAzyme to generate output ssDNA for CRISPR-Cas12a activation. (D) The binding of allosteric transcription factor (aTF) to the target molecule releases output dsDNA for CRISPRCas12a activation.

Recent findings indicate that the cas12a proteins have both trans and cis cleavage activities on ssDNA regardless of the sequence. Notably, Cas12a is the first Cas protein to be identified whose ternary complex has been shown to have trans-ss DNA cleavage ability. Research shows that Cas12a may have acquired single-stranded DNA ability through evolution due to the abundance of viruses in the environment. Thus, gaining a significant role as a powerful and dominating weapon to eliminate invasion by foreign ss DNA. The well-characterized variants of cas12, cas12a, and cas12f, formerly known as cas14 lack the HNH domain but nevertheless, achieved the PAM dependant cleavage with RuvC domain alone. Recent findings have reported Cas13a also called C2Ca, an RNA-guided and RNA-targeting CRISPR effector from the class 2 type VI CRISPR system, was found to have the trans-cleavage activity on RNA. Additionally, the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA [9].

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4. Efficient sensing mechanism of CRISPR/Cas derived biosensors

Electrochemical biosensors register physical-chemical and biological change and possess high throughput of the biological recognition process. Depending on the type of biological recognition, sensors are classified into biocatalytic devices and affinity sensors. Biocatalytic sensors integrate enzymes and whole cells as recognition elements leading to exquisite specificity and a significant rise in the rate of reaction whereas affinity sensors make use of extreme selectivity and specificity for acquiring higher sensitivity. The electrochemical transducer responds to the binding event and converts the electrical response to an output that can be amplified, stored, and displayed [14]. Due to its signal-off architecture, these electrochemical sensors provide limited sensitivity and productivity. To overcome these limitations the CRISPR/Cas12a based electron sensing biosensors have been developed for non-nucleic acid targets. CRISPR/Cas12a-based immobilization-free electrochemical biosensors can detect small molecules and proteins by adjusting regions for target recognition in RCA components [15]. Transcription factors (TFs) assay seems to be path-breaking as it is found to be involved directly in many diseases including cancers. CRISPR/Cas 12a based biosensors for the detection of transcription factors have been developed. The biosensing mechanism is based on the interaction of TF’s with double-stranded DNA activator eliminating Cas12a/crRNA from contacting and interacting with the 14 activators, thus inhibiting Cas12a activation. As a consequence of this strategy, the DNase activity of Cas12a was controlled and several TFs with well-defined binding sites could be quantified at the picomolar level with high precision [16, 17].

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5. Implementation of CRISPR/Cas amplification strategy for protein detection

Recent findings report that the implementation of various nucleic acid amplification strategies led to improvements in analytical specificity and sensitivity and the development of point of care (POC) diagnostics. For example, the best-studied reaction is the amplification employing the Cas9 nickase (Cas9nAR) which when combined with polymerase and primers may substantially duplicate double-stranded DNA (dsDNA) without requiring heat cycling as does the polymerase chain reaction (PCR) [18].

5.1 iPCCA: isothermal proximity CRISPR/Cas 12a assay

In contrast to PCR, isothermal proximity assay seems to be an effective protein quantification assay for disease biomarkers and point of care diagnostics. Recent advances in the CRISPR/Cas technique specifically combining recombinase polymerase activity (RPA) and ssDNAse activity have led to the discovery of a series of isothermal assays for protein quantification. iPCCA relies on proximity binding for target recognition due to which it holds the potential for detecting non-nucleic acid targets such as proteins. However, isothermal amplification does not necessitate the use of advanced and sophisticated thermal cyclers and hence is more commonly used in biosensing [9].

5.2 Aptamer-based assay for femtomolar detection of proteins

The most widely used bioassay, ELISA (enzyme-linked immunosorbent assay) has revolutionized the ability to detect a wide variety of antigens. Complex chemical structure and restricted catalytic efficiency of HRP has a direct correlation with poor sensitivity in picomolar and nanomolar concentration. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious disease. CRISPR/Cas 13a based signal amplification strategy also called CLISA has been used to develop a 10 fold high-sensitive method for detecting low abundance. Recently, CRISPR/Cas13a has been recently demonstrated to have RNA-directed RNA cleavage ability. This RNA-guided trans-endonuclease activity is highly specific, being activated only when the target RNA has the perfect complementary sequence to the crRNA and is highly efficient. This potent signal amplification ability of CRISPR/Cas13a enables the development of direct RNA assays with a sensitivity down to the femtomolar level [19, 20].

5.3 CRISPR/Cas 12a controlled aptasensor for protein detection

Aptamer, a highly selective recognition element has been combined with various analytical techniques to increase the sensitivity of protein assay. Amongst these, an electrochemical technique using specific aptamers as recognition elements exhibits great promise in detecting protein duo to its attractive merits, such as high selectivity and sensitivity, the potential for miniaturization, and ease of integration with additional components [21]. Recent findings have demonstrated that the electrochemical aptasensor has been effectively used for the detection of thrombin in femtomolar concentration. It has been reported that once CRISPR RNA (crRNA)-directed Cas12a binds to a specific target DNA, the conserved RuvC nuclease domain in Cas12a will non-specifically cut single-stranded DNA (ssDNA). A homogeneous electrochemical aptasensor has been reported for sensitive and specific detection of thrombin by utilizing binding-induced DNA strand displacement strategy as the transduction element of thrombin and rolling circle amplification-regulated CRISPR/Cas12a for signal amplification. Importantly, this homogeneous electrochemical aptasensor can detect the femtomolar range of thrombin, and exhibited good specificity relative to other interfering blood-relevant proteins. The BIDSD-RCA-CRISPR/Cas12a is implemented in three steps, but this electrochemical aptasensor dispenses with the need for probe surface-immobilization procedure, simplifying the preparation process, and reducing the operating cost of the analysis. The strategy further could be applied to detect another disease-related protein biomarker in early diagnosis in the future [22].

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6. Anti-CRISPRs: potential repressors of CRISPR/Cas

The struggle for life between bacteria and their infecting viruses (phages) has led to the development of numerous bacterial defense mechanisms and their phage-encoded opponents. Recently, anti-CRISPR proteins have been identified, which inhibits the CRISPR/Cas system. The mechanism by which anti-CRISPR proteins inhibit CRISPR/Cas provides an extensive set of valuable tools to both understand and manipulate CRISPR [21]. Several findings report that the growing number of anti-CRISPR families has a significant impact on CRISPR/Cas function and has been a driving force in the evolution of CRISPR-Cas. These Anti-CRISPR systems rely heavily on Aca proteins due to their extensive interaction with anti-CRISPRs and the presence of Aca genes has the potential to act as anti-anti CRISPR playing a vital role in CRISPR-based antibacterial technologies [22, 23]. Anti-CRISPR ranges from 50 to 150 amino acids with no sequence similarity. Recent finding demonstrated that phage carries atleast one anti-CRISPR gene to avoid elimination by competent hosts. The unique mechanism of anti-CRISPR results in sequence-specific transcriptional repression system. Type II anti-CRISPRs have more evident biotechnological uses, given the widespread usage of CRISPR-Cas9 genome editing tools. Their application could be critical for gene drive and gene therapy technologies [24].

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7. Future perspectives of CRISPR-Cas technology

CRISPR/Cas based technology has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B, or even high cholesterol [25, 26, 27]. It is likely to be many years before CRISPR/Cas technology is used routinely in humans. CRISPR/Cas technology emerged as a versatile technology with wide application in the genome sequence editing, molecular studies of various gene functions, protein detection, gene therapy, and in the biomedical science as a diagnostic technology for detection of covid 19 like viral, bacterial, and various genetic disease [28]. Cancer is one of the fatal diseases that has severely threatened human life and caused a tremendous burden for society [29]. Early diagnosis of cancer is of great benefit to treat patients in early stages which leads to improve the survival rate of cancer patients. In body fluids detection of cancer related biomarkers is a critical kind of noninvasive technique for cancer diagnosis. Nevertheless, existing techniques of cancer biomarker detection always depends on a large-scale instruments and required sophisticated operation [30]. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/Cas) based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, with great promise as the next-generation molecular diagnosis [31]. In the future, genome-wide screening for various genetic disorders, and cancer subtypes should be conducted to identify specific genetic and epigenetic targets for CRISPR technology to be most effective. The functionality of the identified mutations and their related signaling pathways need to be thoroughly analyzed before they are manipulated for therapy with CRISPR technology [32]. More in vivo research on Cas9 epigenetic regulation is needed to better understand its impact on cancer epigenetics. The use of synthetic biology for Cas9 modulation can be further extended to create real-time predictive algorithms for specific metastatic pathways that update as epigenetic regulation progress and the cancer advances so that treatment can always be precisely one step ahead of cancer. Ongoing research has the potential to optimize and advance CRISPR technology, culminating in the clinical realization of its full potential for breast cancer diagnosis, modeling, and treatment [29, 33, 34]. In the future, CRISPR/Cas technology will be used as a unique promising technology to study the various genes for their function, for identification of mutations and their correction, this technology will be used in tumor angiogenesis research for cancer treatment [35], CRISPR technology also used for modification of genetic sequence to develop various organisms for the benefit of human and environmental protection. Much research is still focusing on its use in animal models or isolated human cells, with the aim to eventually use the technology to routinely treat diseases in humans (Figure 4).

Figure 4.

Applications of CRISPR/Cas system in detection of various molecules [36].

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8. Conclusion

From many years scientists have learned about genetics and gene function by studying the effects of alteration in DNA sequence. Artificially by making a change in a gene, either in a cell line or a whole organism, it is possible to study the effect of that change to understand what the function of that gene is. For a long period geneticists used chemicals or radiation to create mutations but this approach is not precise and specific and due to its randomness for several years scientists have been using ‘gene targeting’ to introduce changes in specific places in the genome, by deletion or insertion either whole genes or single bases. Conventional gene targeting has been very valuable for studying genes and genetics, however it takes a long time to create a mutation and is fairly expensive But the CRISPR/Cas9 system based technology currently stands out as the fastest, cheapest and most reliable system for ‘editing’ genes. In the last decade CRISPR/Cas is a genome editing technology that is creating a an atmosphere of excitement in the science world because of its faster, cheaper, promising, precise, sensitive and efficient and more accurate nature than previous conventional techniques of genome engineering and it has a wide range of potential applications. CRISPR/Cas technology have made it possible to edit the genomes of most cell types precisely and efficiently hence (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and applications in almost all branches of life science, biomedicine and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas technology able to detect various targets starting from nucleic acids to proteins. Incorporating CRISPR/Cas systems with numerous nucleic acid amplification strategies allows the generation of amplified detection signals, enrichment of low-abundance molecular targets, enhancements in analytical specificity and sensitivity, and development of point-of-care diagnostic techniques. It is concluded that the CRISPR/Cas systems in association with functional nucleic acids (FNAs) and molecular translators permits the detection of non-nucleic acid targets, like proteins, metal ions, and tiny molecules. Productive integrations of CRISPR technology with nucleic acid amplification techniques lead to sensitive and fast detection of Protein.

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Acknowledgments

The authors would like to thank Dr. B.A. Mehere, Principal and Head of the Department of Biochemistry and Biotechnology, Dr. Ambedkar College, Deekshabhoomi, Nagpur, India, for providing research space and facility.

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Rita Lakkakul and Pradip Hirapure

Submitted: December 14th, 2021Reviewed: January 6th, 2022Published: April 1st, 2022