CRISPR-Cas9 and RNA interference (RNAi) essay

Introduction 

• CRISPR-Cas9 and RNA interference (RNAi) are two revolutionary techniques in genetic manipulation and analysis, each playing pivotal roles in advancing our understanding and control of genetic material. CRISPR-Cas9 functions as a precise gene-editing tool. Recognition of the target sequence in the gene of interest is carried out by designed single guide RNA. RNAi of Post-Transcriptional Gene Silencing is a conserved biological response to double-stranded RNA that can be used to study the function of genes in tumours and to identify new drug targets by effectively silencing the target gene. It achieves this by using small interfering RNA (siRNA). 

CRISPR-Cas9 & RNAi mechanisms and applications 

• CRISPR-Cas9 can be guided to any target sequence adjacent to the protospacer adjacent motif, a short DNA sequence near the 3' terminal of the target sequence that the CRISPR locus is not destroyed by Cas enzymes. Cas9 is a DNA endonuclease that cuts DNA strands at a specific location, creating a gap that can be filled with new DNA. The sequence for Cas9 5'-NGG-3', where N is any nucleobase followed by two guanine nucleobases. CRISPR-Cas9 can be used to correct disease-causing mutations in human cells to treat genetic disorders that are currently untreatable. For example, CRISPR-Cas9 has been used to correct the mutation that causes cystic fibrosis (CF) in intestinal organoids. 

• RNAi involves small, exogenous RNA molecules (siRNA or miRNA) that bind to complementary mRNA sequences, leading to their degradation or translation inhibition small interfering RNA (siRNA). RNAi can be used to inhibit the expression of viral genes, control viral replication, and hinder the assembly of viral particles. For example, RNAi has been used to target the X gene of HBV and to inhibit HIV-encoded RNAs. RNAi is a knockdown technique, meaning that expression of one or more of an organism's genes is reduced. It can be used to target dominant mutant oncogenes and to enhance the effects of existing drugs. It has shown promise in treating diseases like cancer and viral infections. However, challenges remain in delivery methods and potential off-target effects and accessibility of RNAi to less reachable parts of the body and reducing toxic side effects. 

CRISPR-Cas9 and RNAi usage in studies 

• Both CRISPR-Cas9 and RNAi technologies have significantly enhanced our ability to decode and understand genomic information. These tools allow researchers to systematically study gene function on a genome-wide scale.  

• By manipulating genes associated with specific diseases, researchers can create more accurate models for study. CRISPR-Cas9 and RNAi enable high-throughput screening to identify genes involved in various biological processes. By designing a guide RNA, a gene to cut can be chosen and a specific PAM sequence near that gene can be targeted. Gene editing techniques can be used in reverse genetics to identify what phenotypes arise from specific genetic sequences.  RNAi silences genes, so the phenotype that arises from the change can be documented and analysed.  

• CRISPR-Cas9 and RNAi have applications that extend beyond a single species. Cross-species gene function studies: Researchers can study homologous genes across different species to understand evolutionary conservation and divergence.  Xenotransplantation using CRISPR-Cas9 can modify pig organs for potential human transplantation.  

• On March 16, 2024, talented surgeons successfully connected a CRISPR-modified pig kidney provided by eGenesis to Slayman, who had end-stage renal failure and was not a candidate anymore for a transplant from a human donor. CRISPR-Cas9 can remove harmful pig genes that produce sugars that trigger an immune response in humans. CRISPR can also add human genes to improve compatibility. CRISPR-Cas9 can inactivate Porcine endogenous retroviruses that are present in all pig genomes that can cause infection in humans. 

Gene editing concerns 

• The power of CRISPR-Cas9 and RNAi technologies brings significant ethical and societal implications. The potential to make heritable changes to human embryos through human germline editing raises profound ethical questions. There are concerns about biosafety and biosecurity with the potential misuse of these technologies. Questions in equity arise about who will have access to these technologies and their benefits. Minority populations are often underrepresented in genomic databases and clinical research, which could lead to unequal access to gene therapies. Gene editing can lead to biological attack or bioterrorism, the intentional release of viruses, bacteria, or other germs that can sicken or kill people. 

• As these technologies advance, it is crucial to engage in ongoing ethical discussions and develop appropriate regulatory frameworks to ensure responsible use. For example, there is concerns about the environmental impact of the release of genetically modified organisms into the environment as they could have unforeseen consequences. To address these concerns, some recommend increasing diversity in genomic sequencing and improving education and transparency. By building partnerships with communities, developing innovative payment models, and forming public-private partnerships, there can be less concerns regarding fairness.