Targeted gene modulation is an important tool for genome editing. Disrupting gene expression through repression is one of the way to determine the function of genes. RNA interference (RNAi) has been one of the most used tools for a programmed knockdown of mRNAs thus being critical in the understanding of the gene function and regulatory networks. However, the RNAi technology has been undermined by the high rate of false positives which arise from off-target effects 1. These off-targets may be a challenge during whole genome screening. More so, due to RNAi being modulated by proteins found in the cytoplasm, gene repression via this strategy deletes cytoplasm mRNA targets. Due to many limitations demonstrated by RNA interference (RNAi) technology, there are emerging methods that have been generated to be used for programmed genome editing that completely silences DNA: sequence-specific endonucleases such as zinc finger, Transcription activator-like effector (TALE) nucleases and CRISPR (clustered regularly interspaced short palindromic repeats/Cas (CRISPR-associated protein) proteins 1. However, of all these methods, the CRISPR-Cas system emerges as a more efficient and powerful tool for genome screening as compared to RNAi and haploid mutagenesis approaches. The introduction of many transcripts into complex genomes requires a more dynamic DNA-binding protein that can be programmed and can generate an effector complex to switch gene expression on and off. Clustered Regularly Interspaced Short Palindromic Repeats referred to as (CRISPR)/CRISPR-associated (Cas) genes occur naturally in bacteria and archaea. They are alsofound in some plasmids but are mostly found in archaea and about 40% of bacteria. They provide a natural adaptive immune system that initiates mechanisms that protects the bacteria from foreign DNA. CRISPR-Cas adaptive defense entails three steps. Adaptation which involves integration of the protospacer sequences from foreign DNA into the CRISPR loci. Expression of the spacers as specific small interfering CRISPR RNA (crRNA) leads to destruction of complementary DNA within the foreign bacteriophage or plasmid thus defending the bacteria 3. Subsequent transcription of CRISPR locus transcripts leads to production of multiple CRISPR (cr) RNAs that guide complex effector molecules generated by Cas proteins to degrade the homologous invading RNA or DNA. crRNA transcription is initiated by different ways in the different CRISPR-Cas systems. During biogenesis of the multiple crRNAs, endonucleolytic cleavage releases crRNAs from the large CRISPR transcript producing RNAs that have small repeat sequence on both ends of the leader sequence. In most cases, the cleaved crRNA is further catalyzed and processed by cas6 superfamily endoribonucleases in almost all Type III and I CRISPR-Cas systems. The 8-nucleotide cleaved by cas6 superfamily endoribonucleases are maintained in crRNA organism and has been demonstrated to have an important function 4. These spacer sequences are unique and separates clusters of identical repeats and are adjoined to groups of Cas genes. The CRISPR loci is comprised of short sequences that are from the foreign DNA/RNA and are interspaced by copies of short direct repeat sequence of 24-37 bp 4 . Comparison of genomes in bacteria and archaea has shown that short spacer-repeat sequences are placed where the leader region and the first repeat meet. Continuous insertion of foreign nucleic acids leads to repeatedly addition of new spacer units thus increase in length of the CRISPR locus. However, the bacteria contain mechanism that help to reduce and limit their sizes 2. Several CRISPR-Cas systems have been identified and are defined by different crRNA species and Cas proteins. Eleven CRISPR-Cas systems have been reported which comprises of the common Cas2 and Cas1 proteins that contains distinct subtype specific Cas proteins identified with the organism where the proteins were retrieved from. Due to distant relationships, CRISPR-Cas systems has been divided into three broad types of Cas proteins which include type I, II and III. For example, Cse system is a Type I systems but of the E subtype or Type I-E system 4 .These classifications are based on function and structural organization of effector complexes that are involved in crRNA-mediated suppression of invading DNA or RNA. All this three types of CRISPR-Cas systems are directed by short CRISPR RNAs (crRNAs) and they have same capability to cleave the foreign DNA/RNA thus initiating adaptive immunity. For example, in Streptococcus thermophilus the three types work independently of each other in regulation of genes whereby they function with different mechanism to initiate adaptive immunity. Genetic engineering of guided RNAs to targeted genes will be more advantageous than other gene editing tools like zinc finger nucleases (ZFNs) and Effector Nucleases (TALENs) 5. General look of the CRISPR-Cas loci derived from S. thermophiles (Sth). CRISPR loci are encompasses of short direct repeats (black rectangles) separated with specific foreign spacer sequences (colored rectangles). Expression of CRISPR is mediated within upstream leader sequences. Each CRISPR array contains a fused subset of Cas genes (shown as colored boxes).
Previous studies have been conducted to demonstrate that CRISPR-Cas9 system is a more reproducible and specific gene editing tool. Furthermore, CRISPR-Cas9 system has been demonstrated to have many application across many disciplines. For instance, Gilbert et al ., 2013 1 demonstrated how CRISPR-Cas9 is a robust and a more specific gene editing tool whereby they used an inactive Cas9 (dCas9) fusion proteins to bind effector domains to specific DNA sequences to either activate (CRISPRa) expression or supress (CRISPRi) of target RNA/DNA. This fusion protein was mediated by gene-specific sgRNAs. To achieve this, they undertook a thorough screening to test the activity of every sgRNA in detail based on the transcription start sites (TSSs) of 49 genes that regulate cellular susceptibility to ricin . They created genome libraries targeting the 49 genes with 10 sgRNA thus ruling out the off-target effects. They confirmed the activity of these libraries by identifying genes that initiate cell development and response to chimeric cholera/diphtheria fusion toxin (CTx-DTA). Through these experiments, Gilbert et al ., 2013 1 were able to show their CRISPRi/a screening approach as a robust, with reduced cytotoxicity and a more reproducible tool. They were able to demonstrate that transcriptional inactivation is reversible, inducible and can target critical genes in the genome of eukaryotes. They also showed that CRISPRi and CRISPRa had the capabilities to modulate expression levels for endogenous genes along a diverse flexible scale. In this study, it was demonstrated that if CRISPRi reagents are well designed it increases the specificity of CRISPR-Cas9 system 1. In summary, this platform entails dynamic strategies for identifying gene function across diverse transcripts encoded by the human genome.
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Another study performed by Tan et al ., 2013 6 also showed that CRISPR-Cas9 system initiates gene editing and DNA sequence knockout as compared to RNAi technology that reduces posttranscriptional target mRNA. They also demonstrated that CRISPR-Cas9 system is less prone to off-target effects than RNAi technology. Mostly CRISPR-Cas9 system screens are usually done in pooled format. This Pooled vector-based screening system is a vital strategy that entails transducing Cas9 expressing cells with lentiviral constructs cassettes containing single guided RNA (sgRNAs), which are chimera of the trans-activating crRNA (tracrRNA) and CRISPR-Cas9 system CRISPR RNA (crRNA). In their study, they examined synthetic CRISPR systems in relation to arrayed screening. They undertook experiments using abnormal DNA replication as an assay. GMNN a control gene in HCT-116 cells strongly expressing Cas9 were targeted using synthetic CRISPR RNAs and they were able to observe big phenotype change in the transfected cells. Further studies showed complete inactivation of targeted GMNN protein and regulation of GMNN DNA. Synthetic CRISPR RNAs screening that was done against 640 genes that were related to ubiquitin led to identification of known DNA replication modulators. This was also supported by siRNA gene knockdown . It was noted that when CRISPR screening was compared to siRNA screens, CRISPR screening had more hits than siRNA screens statistically. These findings demonstrated that synthetic CRISPR system can be used as a functional arrayed genomic screening tool 6 .
CRISPR-Cas9 system has also been applied in cancer medicine research whereby colorectal cancer has been regulated using CRISPR-Cas9– mediated genetic engineering of human intestinal organoids 7. Human colorectal tumors contains repeatedly mutations in genes that are found in PI3K, TGF-b ,WNT MAPK and TP53 pathways which are important in stem cell signaling. These mutations in the above pathways leads to colorectal cancer but the specific cause of cancer in this mutations is not well defined. Matano et al ., 2013 7 demonstrated that CRISPR-Cas9 system can be used as a genome-editing platform to create mutations in organoids that originate from human intestinal epithelium. To mimic intestinal environment, Isogenic organoids that contains mutations in the tumor suppressor genes TP53, SMAD4 and APC, and in the oncogenes PIK3CA and KRAS were selected . After implantation of engineered Organoids in the kidney subcapsule in mice, the organoids produced tumors but all five mutations grew without depending of niche environmental factors in vitro. These engineered organoids generated micro-metastases that had dormant tumor-causing cells after injection into the spleen of mice which did not colonize in the liver. However, those engineered organoids produced from chromosome-instable human adenomas produced micrometastatic colonies. These findings demonstrated that mutations of genes in stem cell signaling pathway initiates stem cell maintenance in tumor affected environment but more molecular activity such as aneuploidy, epigenetic alterations and copy number variations are needed for metastasising cells 7. Gudbergsdottir et al., 2011 2 challenged CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus to plasmid genes viral and, and protospacers to determine their dynamic properties. During the construction of genes and protospacers, sequences that matched with spacers of CRISPR loci were included and also multiplpe of mismatches were also included. Constructs harboring the protospacers were cloned into expression vectors containing pyrE/pyrF genes and transformed into uracil auxotrophic hosts from Sulfolobus islandicus REY15A or Sulfolobus solfataricus P2. Few transformants were produced and they had either partial deletion of CRISPR loci or complete deletion of the CRISPR/Cas modules. These deletion process did not depend on whether genes were transcribed or not. The availability of the protospacer CC motif was demonstrated to be vital for causing of the deletions in the family I CRISPR loci 2. Furthermore, it has been shown that the CRISPR-Cas9 system can target cellular mRNAs translation 8 . CRISPR-Cas9 system has been recently demonstrated to target viral RNA and this has led to questions whether it can mediate translation of cellular mRNAs. Liu et al ., 2016 8 reported that the first 14 nucleotides in the 5′ end of sgRNA are very vital in the process of translation of cellular mRNAs. It was also demonstrated that CRISPR-Cas9 can repress the protein expression of a non-targeted gene without having an effect on its DNA sequence but lead to unintended phenotypic changes. Designing of RNA aptamer-ligand complexes is very critical to stopping the translation machinery. This results also suggested that CRISPR-Cas9 system should be very specific when it comes to targeting of genes and should be able to avoid off-targets 8. With the above-mentioned applications of the CRISPR-Cas9 system, the technology still has some challenges. One of the main limitation of this technology is the delivery methods used to introduce CRISPR-Cas9 system into the target cells. Delivery of this system in vivo still remains a big limitation of the technology. Physical and viral vector delivery methods have been used to deliver the CRISPR-Cas9 system. The viral vector has been reported to have safety limitations and limited packing capacities. Physical delivery methods have been used to deliver the system in vitro but the delivery of in vivo environment still pose a great challenge. The use of non-viral, lipid and polymer nanocarriers is the future for genome editing in CRISPR-Cas9 system delivery. Another limitation is the specificity of CRISPR-Cas9 system. Lack of specificity leads to Off-targets still poses a big challenge in the development of this technology. Many strategies and computational strategies are being employed to overcome these limitations. However, there is no comprehensive solution to false-target effects although the CRISPR-Cas9 system is a better platform when compared to RNAi screens which are defined with many false positives 8.
In conclusion, gene regulation is a vital tool for genome-programmed editing. Disrupting gene expression through repression is one of the ways to determine the function of genes. CRISPR)/CRISPR-associated (Cas) genes are naturally found in archaea and bacteria. These CRISPR-Cas systems provide a natural adaptive immune system that protects the bacteria from invading DNA. CRISPR-Cas9 system is has been reported as a powerful platform for basic biology research and engineering biology whereby it can be used as potential therapeutics for genome modification. The CRISPR - Cas9 system still has wide range of application in industry, agriculture, and medicine. The system can to mediate genome modifications in both prokaryotic and eukaryotic systems. Fusion of transcriptional activators or repressors to nuclease-deficient Cas9 enables the CRISPR-Cas system to activate or inactivate transcription of the target genes the whole genome. CRISPR-Cas system emerges as a more efficient and a powerful tool for genome screening as compared to RNAi and haploid mutagenesis approaches. Previous studies have been conducted to demonstrate that CRISPR-Cas9 system is a more reproducible and specific gene editing tool. Despite the diverse applications of CRISPR-Cas system, it is still faced by some limitations namely the choice delivery system and off-targets effects. A lot of research is being done to overcome these limitations. Future studies should be focused on detecting potential off-target effects at the protein level in the targeted genes. We need to do more research and testing on sgRNAs to determine if CRISPRi/a is a good approach for understanding gene function and if it is a feasible strategy can it reduce the off-targets effects as compared to other tools. In generally, this platform of switching on and off one or more genes will lead to creation of an important tool for understanding how gene expression is performed in organisms’ genomes.
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