Clustered regularly interspaced short palindromic repeats (CRISPR)

In the past decade, CRISPR came onto the scene and made revolutionary advances in genome editing, and now it has become powerful a tool in different areas of life sciences research. CRISPR-Cas stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated. CRISPR was initially known in bacteria, specifically E. coli, in 1987. It is the adaptive immune system in prokaryotes where small guide RNAs are employed for sequence-specific interference with invading nucleic acids. CRISPR-Cas is made up of a genomic locus dubbed CRISPR that incorporates short repeating elements (repeats) separated by unique sequences (spacers) that can come from mobile genetic elements (MGEs) like bacteriophages, transposons, or plasmids. An AT-rich leader sequence precedes the CRISPR array, which is normally flanked by a set of case genes that code for Cas proteins.

According to the structural composition of the effector genes, the CRISPR-Cas system is split into two classes. The type I, III, and IV CRISPR systems are all part of the class 1 CRISPR system, which consists of multi-subunit effector nuclease complexes. The Class 2 CRISPR-Cas system, which comprises the type II, V, and VI CRISPR-Cas systems, consists of a single effector nuclease, and routine practice of genome editing has been achieved, thanks to the development of the Class 2 CRISPR-Cas system. DNA editing is done with types II and V, while RNA editing is done with type VI. Through the DNA double-strand break (DSB) repair pathway, transposase-dependent DNA integration, base editing, and gene regulation using the CRISPR-d Cas or type VI CRISPR system, CRISPR approaches can produce both quantitative and qualitative changes in gene expression.

In any eukaryotic organism, notably mammals, genetic engineers may effectively harness the CRISPR/Cas system and target genes of interest to modulate their functions. The molecular biology of CRISPR/Cas demonstrates how it can be used to detect disease-causing genetic variants by applying synthetic guide RNAs (gRNAs) and other components to the target region of interest in a DNA molecule for the desired application. CRISPR/Cas9, the most extensively used CRISPR system, usually targets the 5′ of a protospacer adjacent motif (PAM) sequence. They cause double-stranded breaks (DSBs), which can be repaired using one of two DNA repair pathways: homology-directed repair (HDR) or non-homologous end joining (NHEJ). In the presence of a repair template, the HDR process allows for precise gene changes. In the absence of a repair template, however, DSBs are repaired by the NHEJ pathway, which introduces insertions or deletions by editing the DNA region, causing target genes to be disrupted by moving the reading frame.

Instead of the HDR, CRISPR/Cas nucleases-induced DSBs are largely repaired by the efficient eukaryotic cellular NHEJ mechanism. Meanwhile, employing Cas9 nickases can improve indel yields and HDR efficiency by optimizing indel yields at gene loci. By boosting the HDR route through gene silencing or decreasing non-homologous end-joining protein activity, employing small-molecule reagents, or producing proteins, the efficacy of the HDR pathway can be increased. DNA repair proteins have demonstrated promising capabilities in this area, but putting these ideas into practice in vivo is difficult. Furthermore, DSBs in cells caused by DNA repair mechanisms are reported, which result in a variety of undesirable genomic changes such as significant deletions and translocations.

We, at Experiome, offer training in CRISPR-Cas vector designing. We provide bioinformatics support for CRISPR-Cas vector design for any gene of interest.  

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