Applications of CRISPR as a potential therapeutic

Abstract

Genetic disorders and congenital abnormalities are present in 2-5% of births all over the world and can cause up to 50% of all early childhood deaths. The establishment of sophisticated and controlled techniques for customizing DNA manipulation is significant for the therapeutic role in such disorders and further research on them. One such technique is CRISPR that is significant towards optimizing genome editing and therapies, metabolic fluxes as well as artificial genetic systems. CRISPR-Cas9 is a molecular appliance that is applied in the areas of genetic and protein engineering. The CRISPR-CAS system is an integral element of prokaryotic adaptive immunity that allows prokaryotic cells to identify and kill any foreign DNA. The Gene editing property of CRISPR finds various applications like diagnostics and therapeutics in cancer, neurodegenerative disorders, genetic diseases, blindness, etc. This review discusses applications of CRISPR as a therapeutic in various disorders including several genetic diseases (including sickle cell anemia, blindness, thalassemia, cystic fibrosis, hereditary tyrosinemia type I, duchenne muscular dystrophy, mitochondrial disorders), Cancer, Huntington's disease and viral infections (like HIV, COVID, etc.) along with the prospects concerning them. CRISPR-based therapy is also being researched and defined for COVID-19. The related mechanism of CRISPR has been discussed alongside highlighting challenges involved in therapeutic applications of CRISPR.

Introduction

Genetic engineering and its therapeutic interventions have recently come into the spotlight, especially in the research fields. Treating diseases by gene alterations, as well as the power to precisely and purposely modify specific regions of the genetic makeup of an organism has always been an area of keen interest and exploration [1]. Different types of highly precise gene-editing methods are used for treating a wide range of ailments, which include genetic abnormalities as well as various forms of cancer [2]. Genetic editing as a therapy option for numerous disorders has made great development and possesses the capacity to modify the genome of eukaryotes in a very accurate manner [3], [4]. One such tool, the CRISPR/Cas systems, which acts as an effective alternative to the conventionally used techniques for genetic manipulations, can be employed for gene therapies as well [4]. Clustered regularly interspaced short palindromic repeats (CRISPR) were observed in E. coli for the first time [5] followed by reporting in several other bacteria as well [6]. The CRISPR/Cas system is an integral element of prokaryotic adaptive immunity that allows prokaryotic cells to identify and kill any foreign DNA [7]. It was then experimentally found that CRISPR along with its related proteins called Cas proteins are related to foreign viral DNA's adaptive immune system [7]. This technological advancement in the field of genetic engineering works as a molecular appliance for alteration of areas of genetic material i.e. DNA/RNA. As a gene editing method, it is not much challenging, develops faster, and is more accurate than methods like transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs). It recognizes and cleaves foreign DNA/RNA in a sequence-specific fashion. This tool allows one to make very precise changes in any gene sequence by utilizing a small guide RNA, templates that provide the edited sequences, which need to be cut and inserted which can either correct the disease-causing genes, inactivate oncogenes or enhance certain traits.

The recent advancements in gene editing approaches that are commonly employed along with the therapeutic potential of the CRISPR/Cas technique are discussed in Section 2. The applications and mechanism of CRISPR technology to construct gene therapy techniques in various disorders including genetic blood diseases, genetic lung diseases, Cancer, DMD, Huntington's, Blindness, viral diseases like COVID, etc. along with the prospects concerning them have been compiled and presented in sub-headings under the concerned section (Fig. 1).

The CRISPR-Cas system is broadly divided into two classes (Classes 1 and 2), which are subcategorized into six different types, namely, II, III, IV, V, VI, and I. The first class has type I, III, and IV systems that utilize more than one Cas protein in its CRISPR ribonucleoprotein effector nucleases. On the other hand, Class 2 systems incorporating type II, V, and VI utilize a single Cas protein [8]. The former is usually spotted in bacteria and archaea. This makes up to ~90% of all recognized CRISPR-Cas loci. The remaining 10%, which is held by Class 2 CRISPR-Cas systems, is majorly covered in bacteria [9]. They together form the ribonucleoprotein complex, which contains the CRISPR RNA (crRNA) and the Cas protein [10]. The latter system is recognized for its potential to modify specific targets using Cas9 protein and single guide RNA (sgRNA) [11]. Presently, type II systems remain to be the most commonly used protein for genetic modification [12]. It is found in Streptococcus pyogenes (Sp) and utilizes the proteins SpCas9 and Cas9 [13]. Horvath et al. [14] outlined that crRNA has the information to attack a particular DNA sequence. PAM sequence or Protospacer Adjacent Motif sequence recognizes the complement relation between the CRISPR RNA and the target sequence. The complementarity is achieved by the multidomain effector proteins [15].

The defense mechanism associated with the system can be categorized into 3 steps:

• Adaptation or spacer acquisition: This stage is further divided into multiple steps. Firstly, a different unique sequence of the invading MGE, which is also known as protospacer, is adjusted in the CRISPR array, further producing a new element. This step hints at the adaptive nature of the immune system in a way that it makes a memory of the attacker's genetic components [16].

• crRNA biogenesis: The CRISPR array goes through transcription, converting into pre-crRNA, which in turn, turns to mature guide CRISPR RNAs. This enhances and enables immunity.

• Target interference: The last step consists of crRNAs being utilized as a guide to particularly inhibit the attacking nucleic acids.

There is more than one-step to employ the CRISPR-CAS9 tools for therapeutic and diagnostic purposes in genetic and protein studies. As discussed in the above steps, Firstly, an organism is selected to manipulate its genetic material. This is followed by choosing the specific gene. Further, the CRISPR and gRNA selection is followed by the construction of a guide RNA or a single guide RNA via the process of synthesis and cloning. A Cas9 protein is selected as well as delivered to the predefined cell along with sgRNA. Hence, the manipulation is validated.

Two separate RNAs, the crRNA as well as trans-activating RNA (tracrRNA) activate and direct Cas proteins to interact with viral DNA sequences that are eventually glued together [13]. Before the pre-decided genetic material is glued, the Cas9 nuclease develops configurational modifications to the sgRNA (RNA Guide) binding and is guided to its target location [13], [17] The binding affinity is determined by a sequence of twenty nucleotides accompanying the corresponding three nucleotides protospacer pattern (called PAM which consists of an NGG or NAG sequence) [13], [17], [18]. After unwinding the DNA, it then binds to PAM resulting in the generation of a hybrid of DNA and single guide RNA; DSB, i.e. double-stranded break is incorporated in the nuclease section at the target DNA [10], [17].

Host cell reacts to the double-stranded break with two distinct repair methods. Non-homologous end attachment, which is a fallible repair technique, mostly contributes to either insertions or deletions (indel). Such indels might trigger frame-shift mutation, premature stop codons, and even decays arbitrated to target genes by nonsense codons, resulting in depletion of normal functions. On the other hand, homology-guided repair makes use of aided recombination of the donor template of DNA to rebuild the DNA, which was cleaved. This technique may also be utilized for introducing clearly outlined mutations, which can be performed by relocating the modified donor template to the target cells. The intensity of the working of nuclease is decided mainly by the binding or interaction efficacy of Cas9 protein. Modifying the single guide RNA scaffold systematically showed the enhanced structure of scaffold, which is further related to the greater binding efficacy of Cas9 to target DNA molecules [19].

Based on the said mechanism, several therapeutic applications of CRISPR are followed in several diseases, which will be listed in detail in the next few sections.