Genetic editing is a set of technologies that give scientists the ability to change an organism’s DNA. These technologies allow researchers to alter DNA sequences and modify gene function. Genetic editing is also known as gene editing, genome editing, and genome engineering. To better understand the complex world of DNA, scientists have created many genetic editing and genetic manipulation techniques. These techniques have widely varying effects on organisms, from small mutations in a single gene to the addition (or subtraction) of entire genes or sets of genes. They involve many different methodologies, from chemicals to enzymes, to proteins or RNA instructions. Scientists are excited about the potential impact of genetic editing on a wide range of fields.
1. Introduction to Genetic Editing and CRISPR Technology
DNA documentation of genes was first discovered in the mid-1950s and man-made techniques to modify genes and DNA sequences were discovered in the 1970s. The first genetic editing technique, a class of tools called ZFNs, was discovered in the 1990s. Next came TALENs in the 2000s, followed by the revolutionary genetic editing technology, CRISPR, in 2012. These technologies have become the foundation for even more advanced design, adaptation, and subsequent development of newer genetic editing and gene manipulation techniques [1]. The CRISPR-Cas9 system, originally described as a secondary adaptive antiviral strategy found in many microorganisms, looked to be a novel and robust genetic editing tool. CRISPR is an acronym which stands for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA repeat sequences found in nearly all prokarytoes. In the CRISPR system, RNA and protein assemble to search for and disable foreign nucleic acids via complementary base pairing. Once a foreign DNA sequence is recognized, a large, enzyme-like protein, the Cas protein (CRISPR-associated protein), cleaves and degrades the target DNA. In this manner, CRISPR RNA sequences are then created from the foreign DNA sequence and incorporated into the gene sequence catalog in the microorganism. Future infections by the same virus (or plasmid) are then recognized because the foreign crRNA has been copied into the host genome. The RNA and associated Cas protein make up the CRISPR-Cas9 system, which, as molecular scissors, cuts DNA at specific genetic locations [2].
2. Historical Development of CRISPR Technology
It is important to understand the historical background and evolution of CRISPR technology. Since its emergence in science, the advances and breakthroughs made possible in genetic editing with this method have been impressive. First described in [3] in 1993 as a defense mechanism in bacteria, the research popularly associated with CRISPR finally began a quest for applied technology with CRISPR-9. The system of type II CRISPR that contained Cas9 as an effector protein drew the attention of the scientific community with its use of short guide RNA to target any specific DNA strand with high specificity and effortlessly create double-strand breaks. With the delivery of DSB repair precision from this technology, innovative solutions to applications such as medical research, animal and plant manipulation, and even more recently with in vivo studies of methylation sequencing monitoring and precise genome editing of eukaryotic cells with the RNA-guided Cas13 framework [4] arose.
Like every other scientific field, there are many unsolved problems, risks, and validity checks to be conducted regarding any unforeseen effects of using CRISPR along with technical problems, but nonetheless it is astonishing to observe how this natural genome editing tool recognized at around 2012 could transform life sciences and produce significant advancements in under 10 years before entering the industry and public domain.
3. Mechanism of CRISPR-Cas9 System
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) bacterial adaptive immune systems recognize foreign genetic elements and degrade them [5]. As a new gene-editing tool, CRISPR-Cas9 is easy and inexpensive to use, and it is widely used in basic research, medicine, and biopharmaceuticals. Researchers take advantage of the ability of this system to recognize specific sequences of deoxyribonucleic acids (DNAs) to introduce double-stranded breaks into the DNAs of living organisms. This article describes the mechanism by which the CRISPR-Cas9 system recognizes and cleaves target DNA. As a model, the type II-A CRISPR-Cas9 system from Streptococcus pyogenes is presented.
The CRISPR-Cas9 system, a signature example of prokaryotic adaptive immunity, recognizes and destroys foreign genetic elements. The signature features of this defense system in bacteria and archaea include genomic loci with abundant and conserved CRISPR sequences and cas clusters, which are transcribed and processed into short CRISPR RNAs (crRNAs) that direct interference against complementary foreign DNAs [6]. CRISPR-Cas systems can also recognize and destroy RNA viruses, which takes bacteria’s innate immunity a step further. Recently, CRISPR-Cas systems have been used as powerful genome-editing tools in diverse organisms. To maximize the efficiency and fidelity of the CRISPR-Cas system against natural xeno nucleic acid invaders, prokaryotes need to balance two opposing but equally important components, namely specificity and sensitivity. The Cas9-guide RNA (gRNA) complex contains two key components that drive the CRISPR-Cas9 system and initial DNA recognition: the interference proteins Cas9, Csn2, and gRNA. The Cas9-gRNA complex is found in all type II-A to II-D CRISPR-Cas systems in bacteria and archaea and has been recently engineered as a powerful tool for genome editing.
3.1. Components of CRISPR-Cas9 System
The CRISPR/Cas 9 system is composed of sgRNA and Cas9. The Cas9 protein contains two nuclease domains: the RuvC domain, which cleaves non-complementary DNA strands, and the HNH domain, which cleaves complementary DNA strands. The sgRNA includes the trans-activating crispr RNA (tracrRNA) and crispr RNA (crRNA). The crRNA comprises a 20 nt protospacer element and a few additional nucleotides, which are complementary to the tracrRNA. The tracrRNA hybridizes to the crRNA and binds to the Cas9 protein forming the CRISPR–Cas9/sgRNA complex to edit the genome sequences. The Cas9–sgRNA complex unwinds the dsDNA, and the complementary sequence in sgRNA anneals to one of the DNA strands. Upon binding, the endonuclease domains cleave both DNA strands three bases upstream of the protospacer-adjacent motif (PAM) sequence. The target site in DNA includes a protospacer complementary to the 5′-end and 20 nt sequence in sgRNA, and a short PAM bound by Cas9 which is adjacent to the protospacer. sgRNA recognizes a specific sequence in the genome, and Cas9 cleaves the DNA sequence. The Cas9 will not cleave sequence in the absence of a PAM. Different bacterial type II CRISPR systems have different Cas9 proteins. The most commonly used Cas9 is adapted from Streptococcus pyogenes (SpCas9) and identifies the 5′-NGG-3′ sequence on the non-target DNA strand as the PAM [5].
3.2. How CRISPR-Cas9 Targets and Edits Genes
A recombinant plasmid (pMax-Cas9-U6-gRNA) was constructed to express a chimeric Cas9 protein (Nest-Cas9(PP)) activity-less in mammals, together with expression of the gRNA. The hybrid activity of the three rAAVs with distinct single-gRNA plasmids targeted the same genomic locus MuRF1 and co-delivered the three gRNAs targeting MuRF1 and the rAAV-delivered Nest-Cas9(PP) mRNA. In one rAAV injection (25E17 vg/cas9), the results demonstrated the efficient knockout of MuRF1 in GH3 cells (on-target) generating mutant genomic forms consistent with Non-Homologous End-Joining repair. Illumina-sequencing confirmed the high specificity of Mutagenesis, with only one out of ~1,974 bases erroneous converted (off-target), using extremely high generating rAAV viral titers. More importantly, the co-injection of these constructs dramatically, and in an unexpected manner, elevated growth-carrying effects in vivomodels to the engineered AAVs.
The ability to program the system to target virtually any locus of interest, the adaptability to other target sequences, and its easy design made CRISPR/Cas preferred over other gene targeting tools. The simplicity of gRNA design and its less cumbersome preparations also made it easy to launch unlimited genome-scale experiments and place CRISPR/Cas in the hands of non-specialized engineers and scientists. Target genes/scaffolds and site-specific gRNAs for each target locus can be designed by using freely available bioinformatics platforms/sites. A number of free and/or commercial online tools like CRISPOR, Snip-Snap, CRISPR-P, CRISPR-toolkit, E-CRISP, CHOPCHOP, and CRISPR-evo and updated versions of some of them are now available to design highly efficient gRNAs [7].
4. Applications of CRISPR Technology in Biomedical Research
The rapid maturation of clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) as gene editing tools has benefitted research, biotechnology and other applications, including clinic, translational, and practical uses. Several prominent applications of the rapid and cost-effective CRISPR-Cas technology as a pivotal tool in basic and applied biomedical research are examined [7]. The approach of harnessing CRISPR-Cas systems (natural and engineered) to perform genetic manipulations has been widely utilized in various organisms to treat or create a range of diverse disease contexts. Preceding discoveries and recent studies thereby open exciting avenues for advancing the application potential of the CRISPR-Cas technology in next-generation biomedical research plus translational and wider applications in science and technology. A brief overview of the principles, methodologies, progress, and challenges of selected prominent applications of CRISPR-Cas technology are provided. The promise for wider biomedical potential and deeper technological application prospects of CRISPR-Cas technologies are hoped to motivate multifaceted investigations in academia, hospitals, and industries [8].
The application of CRISPR-Cas gene editing technology in basic and applied biomedical research is discussed first, including gene therapy/removal/inhibition, disease treatment/prevention, knockouts of target genes, creation of disease models, etc. The technical principles, methodologies, current progress, and limitations of these applications are examined. Addressing these challenges is desirable and hopeful, so this powerful technology can be employed more widely and extensively in academic basic research, in data collection in animal models, and for validation of preventative/treatment methods in the near future. In-depth investments by interdisciplinary teams are recommended for further developmental approaches for wider future application angles.
4.1. Gene Therapy and Disease Treatment
As a powerful genetic editing technology, CRISPRs open up opportunities to treat inherited genetic diseases. A wide variety of CRIPR/Cas9-based gene therapies are being investigated in experimental stages for animal and cell models of diseases such as heart disease, hypercholesterolemia, muscular dystrophies, diabetes, cystic fibrosis, and hemi-mutations of the fragile X syndrome [9]. Given the difficulty of treatments for such ailments with existing medications or interventions, CRISPR/Cas9 is an attractive powerful tool to explore. For any therapy targeting a disease-causing mutation, the most crucial aspects are finding the best molecular address for targeting and delivering the editing approach to the specific tissue/cells for intervention. Ectopic approaches using lipid nanoparticles, viral vectors, and electroporation may be suitable for somatic tissues. On the other hand, germline editing may involve either engineering reproductive cells directly or expanding a large knock-in population from the edited zygote. In such cases, the long-term genotype stability can be guaranteed, but safety concerning potential off-targets and environmental spread should also be considered.
4.2. Creating Disease Models for Research
Genome editing using CRISPR technology has rapidly progressed since its introduction. Early demonstrations using CRISPR/Cas9 showed that targeted, precise gene editing could be achieved in a wide variety of organisms, including organisms of biomedical and agricultural interest. The rapid advancements of this technology have also led to its applications in creating disease models for research. These applications of the CRISPR/Cas9 technology have focused primarily on genetic diseases, protein-misfolding diseases, and infectious diseases. The creation of these disease models is an important part of efforts to systematically and thoroughly understand the relationship of whole genomes to pertinent phenotypes in a wide variety of organisms. This chapter highlights the recent contributions of CRISPR technologies to the creation of disease models [9] [10].
5. Ethical Considerations and Controversies in Genetic Editing
Understanding genetic editing is not only about the transmission and modification of DNA sequences via cellular machinery. It is also important to be acquainted with the ethical considerations and the controversies it generates in the wider society, especially in the wake of the development and implementation of CRISPR technology. The broader consequences of a scientific technique can often be more far-reaching than the technique itself, and CRISPR technology is no exception [11].
The attention surrounding the practical applications of CRISPR technology rests on the extraordinarily diverse range of organisms that can be genetically engineered, and the high level of efficacy and alignment between expectation and achievement. However, public mistrust toward the implementation of a new technology usually manifests itself as a blame-game between stakeholder groups that are unsure how to respond to unforeseen consequences, and the market. In addition, sociologists, business investigators, and scholars from the field of science and technology studies have pointed out that public distrust, often expressed as fears, concerns, and speculations regarding unforeseen adverse consequences, is common in rapidly changing socio-technical systems [12].
6. Recent Breakthroughs and Innovations in CRISPR Technology
Despite CRISPR-Cas9 being the most well-known and publicized type of CRISPR genome editing, scientists have been hard at work on alternate and improved versions of CRISPR genome editing. The most recent advance in CRISPR technology is “Prime Editing,” developed by researchers from the Broad Institute, MIT, and Harvard in 2019. Prime editors can not only deletions and insertions of DNA sequences, but they can also make specific point mutations by transcribing a new DNA sequence using reverse transcription, which means that more diseases can be targeted for editing. There is also the possibility that off-target editing will be minimized compared to other Cas9-based CRISPR genome editing systems [13].
There are also additional interesting recent advances in CRISPR technology. A new CRISPR system called C2c2, or Cas13, has been developed, which recognizes RNA instead of DNA. C2c2 was discovered in the bacterium Leptotrichia wadei and can be used to target RNAs from viruses or non-time critical knockdowns of genes by engineering guide RNAs (gRNAs) and expressing them with Cas13. CRISPR technologies have also been combined with transcription factor decoys to upregulate or downregulate the expression of genes by sequestering transcription factors, the expression of genes can be controlled, or enhancer elements can be analyzed. CRISPR systems have even been optimized for use in Plasmodium falciparum, the deadliest malaria parasite. CRISPR variants can be used to screen for essential genes and tagging for localization studies [14].
6.1. Prime Editing
To better manage and combat various human diseases including genetic disorders, cancer, and infectious diseases, new genome engineering methods and tools are continuously being developed. The CRISPR technology, which emerged a decade or so ago, has revolutionized genome editing research. Its impact began to be seen in many research areas like genomics research, epigenetics research, gene expression analysis, and transcriptome analysis. Recently, breakthrough innovations in the CRISPR technology have allowed genome editing applications to be used at precision levels, expanding its impact in biomedicine, agriculture, and environmental modifications [15]. Among these innovations, prime editing has been recognized as the ‘next-generation CRISPR technology’. First published in 2019, prime editing opens up a new dimension in precision genetic editing—no double-strand breaks, no easy alternative repair pathways for unwanted modification, no donor template required for directed insertion, and no design restrictions regarding the type of edits. Though still at the infancy stage, genomic prime editors for many plants, animals, and fungi have already been developed or are in progress [16].
Understanding prime editing in the context of CRISPR technology, and agricultural or medical genome-related research in the context of prime editing, is fundamental to grasping the forefront of genetic editing technology.
6.2. CRISPR-Cas Systems Beyond Cas9
Employing CRISPR-Cas9 as a gene-editing tool is only the tip of the iceberg. Emerging innovations with existing CRISPR-Cas9 systems, Cas proteins, and other systems are on the horizon and will diversify the capabilities of CRISPR-Cas applications. Continued exploration of existing and novel Cas proteins, other CRISPR systems, and novel components must be at the forefront of CRISPR-Cas research beyond Cas9 to continue fueling this field’s rapid expansion.
There exist a number of recently identified CRISPR-Cas proteins or systems that are not type II CRISPR-Cas systems, such as the recently identified type V-A Cas12 and type VI-A Cas13 systems [17] [7]. The type V-A systems are surprisingly simple compared with the type II systems (i.e., Cas9 systems) in that most currently described type V-A systems consist solely of Cas12 (i.e., C2c1) protein and crRNA. Cas12 proteins are effectors that combine target DNA recognition, crRNA interaction and foreign DNA cleavage within a single protein. RNA-dependent DNA cleavage by the type V-A system, while leading to similar final outcomes as the type II systems, can involve LwaCas12a collateral cleavage on DNA, unlike the RNA-dependent DNA cleavage mechanism that requires the assistance of a second protein in type II. The type VI-A systems are crRNA-guided RNA-targeting systems composed solely of Cas13 proteins.
7. Challenges and Limitations of CRISPR Technology
CRISPR technology is one of the most attractive and powerful tools for genetic editing available today. However, despite the success and rapid advances made in this technology over a relatively short time frame, many challenges and limitations still remain [18]. Such challenges need to be fully recognized to appreciate the current limits of CRISPR, raise the awareness of its shortcomings and provide guidance on future avenues of research in this field.
Realizing the full potential of CRISPR technology will require research across various disciplines including molecular biology, genomics, bioinformatics, proteomics, transcriptomics, metabolomics, and engineering. Such an effort involves both basic science and translational research. Nonetheless, apart from very exciting applications for testing the “proof of principle” for CRISPR, there will be challenges that need to be addressed to deploy the sophisticated CRISPR tools across thousands of scientific laboratories internationally. The DNA repair mechanisms that are recruited after Cas9 cleavage to repair the NHEJ do not appear to be uniform and vary from target to target, with a range of editing efficiencies. The generation of both mutant alleles and the subsequent recovery of almost an equal number of precision edited alleles presents a specific challenge that will need to be fully understood to produce user-friendly CRISPR tools [19].
8. Future Directions and Potential Impact of Genetic Editing with CRISPR
With rapid progress made in the CRISPR technology arena, future directions and implications should be highlighted. In the near future, the application of base editors and prime editors in human trials will be very exciting to watch, because many genetic disorders are caused by point mutations, which can be corrected by new types of genome editors with very low levels of unintended genomic alterations [1]. Basic researchers are striving to increase the editing efficiency of base editors and prime editors, and a striking increase in the number of clinical trials involving CRISPR-based genome editing with these new tools is expected.
Additionally, another exciting possibility is on the horizon: results from the first studies attempting to treat common, non-Mendelian disorders with CRISPR-based genome editing. These studies attempt to prevent the development or progression of multifactorial diseases by targeting genes with critical roles in disease pathophysiology. For instance, interleukin-1β and MMP13 were ablated to prevent the progression of osteoarthritis in surgically induced mouse models. Angiotensinogen was deleted to prevent the development of hypertension in spontaneous hypertensive rats. The NLRP3 inflammasome was disrupted to alleviate common inflammatory skin disorders in mouse models. Finally, beta-secretase 1 was targeted to suppress cognitive deficits in mouse models of Alzheimer’s disease. Although there is still very much to be developed, targeting just the right genes that can inhibit pathologic pathways might provide effective treatments for chronic disorders [14]. Recently, important technologies necessary for viable CRISPR-based therapeutics—safe and scalable production of lipid nanoparticles for mRNA delivery and the generation of multiplex gRNA libraries using genetic engineering—have been established, demonstrating the maturation of the CRISPR field suitable for applications in large mammals. Considering the positive outlook of the industry’s growth and enthusiasm in the academic/commercial arena, extensive research in the CRISPR-based genome editing field brings cures for human genetic diseases closer to being in our grasp.
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