CRISPR/Cas9: BIOLOGY, MECHANISM OF ACTION AND CHALLENGES

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INTRODUCTION | CRISPR/Cas technology

CRISPR/Cas9 technique has not only shown tremendous growth in scientific research but it has also drawn much attention as a promising gene editing tool for cancer, genetic disorders, and disease-causing bacteria. Out of 13469 articles that show the word “CRISPR” as Best Match, around 5665 articles have been published in PubMed from 2018 to till now.

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The burgeoning field of CRISPR/Cas technology has surpassed other gene editing tools due to its ease to handle and low cost.

CRISPR is the short form of the name “Clustered Regularly Interspaced Short Palindromic Repeats”. The CRISPR Associated protein is shortened as a Cas protein. Therefore the technique is named as CRISPR/Cas technology in short.

The CRISPR/Cas systems naturally occur in bacteriaand archaea (Prokaryotes) to acquire immunity against viral infections and plasmids. Later, scientists started to adopt this system to edit genes in prokaryotes as well as eukaryotes.

HISTORY

The first sign of the CRISPR/Cas system was discovered by a Japanese research group in 1987. They identified a pattern of short repeat sequences interspersed with short, non-repetitive “spacers” in Escherichia coli genome.  In 2012, two scientists (Doudna and Charpentier)   programmed the CRISPR/Cas9 system to cleave specific DNA sequences. This lead to emerge CRISPR/Cas9 as a promising gene editing tool.

BIOLOGY

The CRISPR/Cas system can be classified into two classes as Class1 and Class2. Class 1 CRISPR/Cas systems employ multi Cas protein complex while Class2 CRISPR/Cas systems accomplish single Cas protein. Further two Classes are divided into six types based on the presence of specific signature genes.

The widely used CRISPR/Cas9 technique belongs to the Class 2 type II CRISPR/Cas system. The CRISPR/Cas9 system naturally occurs in Streptococcus pyogenes bacterium as an adaptive immune system to disrupt virus and plasmid which invade bacteria. In this system, a short sequence of foreign DNA (virus or plasmid) is integrated into the bacterium genome to create “identity” to recognize similar invasions prior to their infections in the future. Once a similar virus invades, the bacteria encode complementary ribonucleic acid strand (RNA) of “identity” which can bind to the complementary DNA sequence of the virus and then cleave it.

The CRISPR/Cas9 system consists of three components: crRNA (CRISPR RNA), tracrRNA (Transactivating CRISPR RNA) and Cas9 protein. This Cas9 protein shows helicase activity (unwind DNA double-strand) as well as nuclease activity (cleave DNA strand).In the bacterial CRISPR/Cas9 system, guide RNA (gRNAà crRNA + tracrRNA) directs Cas9 protein which functions as a DNA endonuclease enzyme to cleave viral DNA strands.

The burgeoning field of CRISPR/Cas technology has surpassed other gene editing tools due to its ease to handle and low cost.

          Figure 1: Classification of CRISPR/Cas System

Instead of two separate RNA molecules, researchers have synthesized one RNA molecule (sgRNA- single guide RNA) which can be used as a similar molecule to crRNA+tracrRNA.It has simplified three component CRISPR/Cas9 system to the two-component system (sgRNA+Cas9).

MECHANISM OF ACTION

The defense mechanism of the CRISPR/Cas9 system in bacteria can be divided into three phases:

  •  Spacer acquisition
  •  crRNA processing
  •  Interference

Each phase is described below with a diagram.  

  • Spacer acquisition

After the viral infection, cas operon produces (transcription followed by translation) cas1-cas2 protein complex which can identify protospacer/spacer sequence (“identity”) of viral DNA and integrates it to the CRISPR array flanked by repeat sequences.  

Later, the tracr gene and CRISPR array transcribe (convert gene into respective RNA molecule) tracrRNA and pre-crRNA respectively whereas the cas9 gene transcribes and translates (convert RNA molecule into respective protein) into cas9 protein.

The burgeoning field of CRISPR/Cas technology has surpassed other gene editing tools due to its ease to handle and low cost.

          Figure 2: Spacer acquisition of CRISPR/Cas9 system

(ii) crRNA processing

The tracrRNA anneals to the pre-crRNA repeat. Then tracrRNA: pre-crRNA duplex binds to cas9 protein. Further, the complex recruits RNaseIII enzyme that cleaves the pre-crRNA at the repeat. Finally, an unknown nuclease trims 5’ repeat-derived portion of the crRNA by leaving 20 nucleotide long spacer sequence for the final phase. This forms the final CRISPR/Cas9 complex for interference.

The burgeoning field of CRISPR/Cas technology has surpassed other gene editing tools due to its ease to handle and low cost.

              Figure 3: crRNA processing of CRISPR/Cas9 System

(iii) Interference

The Cas9 of the effector complex identifies target DNA sequence through PAM (Protospacer Adjacent Motif) recognition that locates in the non-target DNA strand (The PAM sequence which recognized by Streptococcus pyogenes is 5’NGG 3′). The 20 nucleotides long, spacer sequence of mature crRNA binds to the target DNA strand in viral DNA. It allows the cas9 protein to activate its two nuclease domains, HNH and RuvC. These two domains HNH and RuvC cleave target DNA strand and non-target DNA strand of virus respectively, by generating a blunt, double-strand break, 3 base pairs front of the PAM. This leads to the destruction of the viral genome.

{5’NGG 3’àN- any nucleotide (adenine/cytosine/guanine/thymine), G- guanine nucleotide}

The burgeoning field of CRISPR/Cas technology has surpassed other gene editing tools due to its ease to handle and low cost.

                Figure 4: Interference of CRISPR/Cas9 System

Bacteria employ the CRISPR/Cas9 system to protect them from invasions. Nevertheless, the same system can be harnessed to edit mammalian genome including humans. Unlike bacteria, it gives rise to double-strand breaks in DNA which are later repaired either by non-homologous end joining or homology-directed repair mechanism in mammalian cells. This garnered much attention from the scientific community to be employed in this state of the art technique to eradicate human diseases such as genetic blood disorders, neurodegenerative diseases, and cancers. Up to date, several clinical trials have been recruited to employ the CRISPR/Cas9 technique to treat cancer and beta thalassemia according to ClinicalTrials.gov database.

CHALLENGES

Even though the CRISPR/Cas9 system holds immense promise to treat human diseases, it faces technical challenges.

The CRISPR/Cas9 system naturally occurs in Streptococcus pyogene bacterium which is harmful to human health. Once it delivers to human cells, the human body can create immunogenicity (provoking an immune response in the human body by substance) which makes the CRISPR/Cas9 system fails inside the body.

Researchers often employ Adeno associated virus vectors (AAV) to deliver the CRISPR/Cas system in vivo. As Cas9 protein is large in size, packaging into the AAV vector is a real challenge. Therefore researchers should either discover alternative vectors which have low immunogenicity or cas9 variants which are smaller in size for packaging.

One more issue is that the CRISPR/Cas9 system causes off-target effects by cutting the wrong piece of DNA. Therefore, it is practical to discover cas9 variants that have broad PAM compatibility and high DNA specificity to avoid this obstacle.

Further, this powerful technique should be employed responsibly and carefully not only to benefit all humankind but also to avoid misconduct which can lead to severe ethical issues in human society.

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Further Reading

  1. Hille, F., Richter, H., Wong, S.P., Bratovič, M., Ressel, S., Charpentier, E. (2018) The Biology of CRISPR-Cas: Backward and Forward. Cell 172(6):1239-1259. doi: 10.1016/j.cell.2017.11.032.
  • Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816-21. doi: 10.1126/science.1225829.

Author

Padmika Wadanambi

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