Introduction:
Genome editing, also known as insertion, deletion, and replacement of DNA, alters genomic DNA at a specific target site in various cell types and organisms. This results in the inactivation of target genes, the acquisition of novel genetic traits, and the correction of pathogenic gene mutations.
Genome editing technology has recently emerged as the most effective way to research gene function, investigate the pathophysiology of genetic disorders, create new gene therapy targets, breed crop varieties, and other topics due to the rapid expansion of the life sciences.
Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nucleases systems are the three most common genome editing methods available today. CRISPR-Cas systems have become the most popular genome editing tool in molecular biology labs worldwide because of the benefits like straightforward design, low cost, high efficiency, strong reproducibility, and quick cycle times.
What Is CRISPR-Cas Systems?
Most bacteria and archaea have CRISPR-Cas, an adaptive immune system that protects them against phages, viruses, and other foreign genetic material. It comprises CRISPR repeat-spacer arrays, which may be further translated into CRISPR RNA and trans-activating CRISPR RNA, and a group of CRISPR-associated (cas) genes, which create Cas proteins with endonuclease activity. When foreign genetic elements penetrate prokaryotes, Cas proteins can break the invaders' DNA into brief bits, subsequently incorporated into the CRISPR array as new spacers. When the same invader returns, crRNA immediately detects it and links with the foreign DNA, directing the Cas protein to break specific regions of alien DNA and protect the host.
What Is CRISPR-Cas Systems Classification?
Classification of CRISPR-Cas systems includes two classes (Class 1 and Class 2), six types (I to VI), and a number of subtypes. Class 1 systems (Types I, III, and IV) include multi-Cas protein effector complexes, whereas Class 2 systems only have a single effector protein (Type II, V, and VI).
One of the most well-known and often utilized types of CRISPR-Cas systems is Type II, derived from Streptococcus pyogenes (SpCas9). The single-guide RNA and RNA-guided Cas9 endonuclease are the two primary elements of the CRISPR-Cas9 system (sgRNA). The HNH and RuvC nuclease domains of the Cas9 protein each cut one strand of the target double-stranded DNA. Single-guide RNAs (sgRNAs) are a condensed form of tracrRNA and crRNA. A Cas9 ribonucleoprotein (RNP), which can bind to and cleave the particular DNA target, is created by the Cas9 nuclease and sgRNA. Moreover, the Cas9 protein needs an adjacent protospacer motif (PAM) sequence to attach to the target DNA.
What Are the Advancements or Breakthroughs Associated With Crispr-Cas Systems?
Since its ability to alter DNA was demonstrated in 2012, CRISPR-Cas systems have become the most popular genome editing technology in molecular biology labs. Many breakthroughs have been made in eradicating harmful mutations, finding essential genes for cancer immunotherapy, and resolving significant issues in organ xenotransplantation. Sadly, specific problems with CRISPR-Cas systems still need to be resolved, including the possibility of off-target effects, the restricted genome-targeting range caused by PAM sequences, and low efficiency and specificity. As a result, several research groups have been working to enhance this instrument.
Dead-Cas9 System
A nuclease dead Cas9 (dCas9) was created by inserting the two point mutations H840A and D10A into the HNH and RuvC nuclease domains. DNA cleavage activity of the dCas9 is absent, while DNA binding activity is unaffected. After that, the CRISPR-dCas9 system may be utilized to either activate (CRISPRa) or inhibit (CRISPRi) the transcription of target genes by joining transcriptional activators or repressors to dCas9. Moreover, dCas9 may be coupled to several effector domains, enabling the recruitment of fluorescent proteins for genome imaging and epigenetic modifiers for epigenetic modification based on the sequence. Also, this technology is simple to use and allows simultaneous regulation of several genes in a cell.
Base Editing System
Base editing systems incorporating dCas9 linked with cytosine deaminase (cytidine base editor, CBE) or adenosine deaminase (adenine base editor, ABE) have been created to increase the effectiveness of site-directed mutagenesis. Without causing double-stranded DNA breakage, it can insert point mutations such as CG to TA or AT to GC into the editing window of the sgRNA target sites. The outcomes of gene mutation are more predictable because base editing technologies significantly reduce the creation of random insertions or deletions. Base editing technologies are not appropriate for all target sequences in the genome due to the limitation of the base editing window. As a result, C-rich sequences, for instance, would result in many off-target mutations. To get around this problem, researchers have always worked to create and improve unique base editing techniques. For effective site-directed mutagenesis, base editing techniques are currently being employed extensively in various cell lines, human embryos, microbes, plants, and animals.
CAS9 Variant System
For the Cas9 protein to recognize and cleave the target gene, there must be an NGG PAM at the 3′ ends of the target DNA region. Other PAM sites, such as NGA and NAG, exist in addition to the traditional NGG PAM sites. However, their effectiveness for genome editing is not very significant. The number of targetable genomic locations is significantly constrained because such PAM sites are only present in around one-sixteenth of the human genome. Many Cas9 variations have been created to increase PAM compatibility for this reason.
These Cas9 variations have allowed scientists to correct several disease-related genomic mutations that were previously unreachable. Nevertheless, These variations have shortcomings, such as poor efficiency and cleavage activity.
What Role Do Crispr-Cas Systems Play in Developing Cell and Animal Models of Human Diseases?
CRISPR-Cas systems have so far been widely used in a wide range of species, including bacteria, yeast, tobacco, Arabidopsis, sorghum, rice, Caenorhabditis elegans, Drosophila, zebrafish, Xenopus laevis, mouse, rat, rabbit, dog, sheep, pig, and monkey, as well as different human cell lines, such as tumor cells, adult cells, and stem cells. The most significant use of CRISPR-Cas systems in the medical profession is the establishment of genetically altered animal and cell models of several human illnesses, including gene knockout models, exogenous gene knock-in models, and site-directed mutagenesis models.
Creating Disease-Related Animal Models for Humans
Understanding gene function, examining the pathophysiology of human illnesses, and creating new medications all require animal models. The use of animal models in fundamental medical research and preclinical investigations is severely constrained by the complexity, expense, and time required by existing methods for producing animal models. Since developing CRISPR-Cas systems, several genetically altered animal models have been created successfully and exceptionally effectively.
Creating Disease-Specific Cell Models for Humans
The use of genetically modified cell models can significantly reduce the amount of time spent conducting research in medical studies because it has been discovered that CRISPR-Cas-mediated genome editing is more effective in vitro than in vivo. For the time being, scientists have employed CRISPR-Cas systems to carry out genetic modifications on different cell lines, such as tumor cells, adult cells, and stem cells, to imitate several human disorders.
What Are the Applications of Crispr-Cas Systems in Disease Diagnosis?
CRISPR-based molecular diagnostic technology has advanced with developing CRISPR-Cas systems and identifying new Cas enzymes (Cas12, Cas13, etc.).
Contrary to Cas9, the Cas13 enzymes have a "collateral cleavage" activity that can cause the cleavage of neighboring non-target RNAs after cleaving the target sequence. Using Cas13's "collateral cleavage" activity as a foundation, the SHERLOCK in vitro nucleic acid detection technology was created (Specific High Sensitivity Enzymatic Reporter UnLOCKing). It comprises fluorescent RNA reporters, sgRNA targeting specific RNA sequences, and Cas13a. To achieve the goal of diagnosis, the Cas13a protein first detects and cleaves the target RNA. It then cuts the report RNA and releases the detectable fluorescence signal.
Conclusion
The development of CRISPR-Cas-mediated genome editing techniques is seen as a significant turning point for molecular biology in the twenty-first century since they have made it feasible and adaptable to change, control, and view genomes. The extensive use of CRISPR-Cas systems in gene function research, human gene therapy, targeted medication development, the creation of animal models, and livestock breeding thus far demonstrate their immense promise for future advancement.
