CRISPR Definition

The acronym CRISPR refers to grouped, consistently interspaced small palindromic repeats that are present in the majority of archaea and bacteria. CRISPR as well as CRISPR-associated proteins (Cas) provide an adaptive immune system capable of defending against viruses, plasmids, and transposons. The CRISPR/Cas9 system also acts as the basis for a genome-editing tool, which may be utilised to modify genes precisely and consistently.

Features of CRISPR Loci

CRISPR loci have an AT-rich leader sequence accompanied by a number of short nucleotide repeats separated by spacer sections. Others are asymmetric and projected to produce unstructured RNA. Within a CRISPR locus, repetitions are conserved while spacers are very changeable and relate to DNA derived from viruses and plasmids. CRISPR repeats and spacers vary from 23 to 55 base pairs in length, with spacers ranging from 21 to 72 base pairs in length.

CRISPR arrays are generally located near clusters of Cas genes. Cas genes encode a variety of proteins, including helicases, nucleases, polymerases, and nucleotide-binding proteins with functional domains. Cas’s proteins participate in the acquisition of spacer DNA, the processing of RNA, as well as the binding and cleavage of targets. CRISPR loci have been identified in the genomes of 90% of archaea and 40% of bacteria, and the genome of an organism may include one or more CRISPR loci (up to 18 have been observed in a single organism).

CRISPR Classes and Types

CRISPR/Cas systems are divided into two categories: Class 1 and Class 2. To break foreign DNA, Class 1 systems employ numerous Cas proteins, while Class 2 systems use a single Cas protein. Types I, III, and IV are found in Class 1, whereas types II, V, and VI are found in Class 2. Types are identified by a gene that is found solely in that type, as well as any other Cas genes that are present. There are 19 subtypes within each kind.

Function and Mechanism of CRISPR in Prokaryotes

CRISPR gives protection against invasive genetic elements to prokaryotes. These loci use genetic material obtained from viruses and plasmids to sequence-specifically target foreign genetic elements. Acquiring spacer DNA from the invading virus, synthesis of CRISPR RNA (crRNA), which enables detection of foreign DNA, and interference, in which the invasive DNA is recognised and cut, are the stages involved in guarding against foreign genetic elements utilising the CRISPR/Cas system.

Acquisition of Spacer DNA

When a virus infects a prokaryotic cell, portions of the viral DNA are extracted and incorporated into the CRISPR locus’ spacer regions. The nuclease enzymes Cas1 and Cas2 are involved in spacer acquisition in E. coli and possibly other CRISPR/Cas systems, given they are the only Cas proteins shown to be conserved across all systems.

PAMs are essential for the recognition of foreign nucleic acids in type I and type II systems. The DNA collected and incorporated into CRISPR loci as spacers may be positioned close to protospacer adjacent motifs (PAMs) in the viral genome. Although additional spacers are normally placed after the leader sequence, they may alternatively be inserted at random into the repeat-spacer array.

crRNA Biogenesis

The CRISPR sequence gets translated and converted into minute interfering crRNAs once foreign DNA is incorporated into a CRISPR locus. The repeat-spacer array is first translated into a single transcript, which is then processed into crRNAs by Cas proteins, which act as a reference for targeting foreign nucleic acids.

CrRNA biogenesis varies amongst CRISPR systems. Cas’s proteins cleave at the edges of stem-loops in lengthy transcripts, resulting in smaller crRNAs. In type II systems, a trans-activating crRNA (tracrRNA) that is equivalent to the CRISPR repeats leads to the synthesis of double-stranded RNA (dsRNA). RNaseIII generates crRNAs by cleaving dsRNA.

Interference

During the interference stage, CrRNAs combine with Cas proteins to form a complex that identifies, targets, and destroys viral genetic material. The crRNA forms base pairs with the corresponding sequence of the viral DNA, indicating that it should be destroyed. It’s possible that identification of the PAM sequence is also essential for foreign DNA recognition. Cas9 performs the interference step in type II systems by employing both a crRNA and a tracrRNA to detect foreign DNA. Cas9 is an endonuclease that, in addition to recognising target sequences, cleaves foreign DNA.

CRISPR/Cas9 System as a Genome Editing Tool

As a molecular biology method, the CRISPR/Cas system has been utilised to modify specific genomes. The genome is modified using the CRISPR/Cas9 technique. Cas9 is a DNA endonuclease that uses RNA as a guide to target and cleave DNA. This technique was streamlined for genome editing by merging crRNA and trans-activating CRISPR RNA (tracrRNA) into a single guide RNA.

The CRISPR/Cas9 system, which is utilised to repair DNA through non-homologous end joining or homology-guided repair, might incorporate a repair template. By modifying the sequence of the guide RNA, the CRISPR/Cas9 system may target any DNA sequence and knock it down, activate it, or change it. Components of the CRISPR/Cas9 system are often included in plasmids that are used to transfect cells and modify their genomes. Multiple changes may be made at once; in one case, 62 genes were altered simultaneously.

Gene Activation and Repression

CRISPR with no nuclease activity (dCas9) may be used to either activate or repress a sequence. Because it lacks the capability to cut DNA, the CRISPR/Cas9 system can target genes without cleaving them. In bacterial cells, Cas9 without nuclease function stops transcription alone; additional proteins are introduced to mammalian cells. Cas9 may also be linked to transcription factors, enabling target genes to be activated.

Altering the Genetic Sequence

A DNA repair template may be put into the CRISPR/Cas9 system, which allows this DNA sequence to be integrated into the correct place. The split DNA fragment is not the only thing that the repair template can fix. Following the introduction of the DNA break, the cell’s DNA repair machinery uses the template to repair the DNA, changing the sequence of the DNA irrevocably.

CRISPR Applications

CRISPR technology, amongst other cell types and species, can be used to make precise genetic modifications in mice, plants, fish, and human cells. CRISPR may be used to construct illness models in animals by knocking down or targeting a particular gene. CRISPR may also be employed to make disease-causing cell lines and study the disease’s underlying mechanisms.

CRISPRs have therapeutic potential as well; they have been authorised for clinical trials in the treatment of cancer and may be useful in blocking the reproduction of viruses such as the herpes virus in humans. They’ve also been used to treat genetic diseases in animals, and they might be useful for limiting the reproduction of viruses like the herpes virus in people. In addition to pharmaceutical applications, CRISPR might be used to create biofuel-producing yeast strains and food crops.

Question and Answer

1.The CRISPR/Cas system provides adaptive immunity against foreign genetic elements in what types of organisms?

1. Bacteria
2. Archaea
3. Eukaryotes
4. A and B

D is correct. CRISPR loci are found in ~40% of bacteria and ~90% of archaea, where they provide the organism with acquired immunity against viruses and plasmids.

2.What does Cas9 do after binding DNA that is complementary to its guide RNA?

1. Replicates it
2. Cuts it
3. Transcribes it
4. All of the above

B is correct. Cas9 is a DNA endonucleases that cleaves DNA it detects to be complementary to its guide RNA.

3. Which of the following is not a feature of CRISPR?

1. Contain palindromic DNA repeats
2. Have highly conserved spacer regions
3. Provide adaptive immunity to Bacteria and Archaea
4. Used as a molecular biology tool for genome editing

B is correct. CRISPR loci contain spacer regions that are not conserved. Conversely, they are highly variable, and correspond to invasive genetic elements acquired from viruses and plasmids.

References

• Chen, K.Y. and Knoepfler, P.S. (2016). “To CRISPR and beyond: the evolution of genome editing in stem cells.” Regenerative Medicine. 11(8):801-816.
• Doetschman, T. and Georgieva, T. (2017). “Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.” Circulation Research. 120(5):876-894.
• Patterson, A.G., Yevstigneyeva, M.S., and Fineran, P.C. (2017). “Regulation of CRISPR-Cas adaptive immune systems”. Current Opinion in Microbiology. 37:1-7.
• Salsman, J. and Dellaire, G. (2017). “Precision genome editing in the CRISPR era.” Biochemistry and Cell Biology. 95(2):187-201.