INTRODUCTION
Many technologies have been developed to manipulate DNA sequences according to our interests. But introducing site-specific modifications in the genomes of cells and organisms was cumbersome. However, transformative phase started with the discovery of genome engineering using RNA-programmable CRISPR Cas9. CRISPR system was discovered as a microbial adaptive immune system. But its conversion into a gene modification tool has enabled precise editing of DNA sequences, finding many applications in agriculture, biofuels and medicinal strategies.
But the Double Stranded Break approach of CRISPR/Cas9 affects its potential in therapeutical uses. Whereas Prime editor- the new genome editor offers precision and versatility because of its asset of constructing only a single stranded break in the DNA sequence. Full evaluation of this method with enormous potency has to be done before bringing it to human practical use.
1. CRISPR/Cas TECHNOLOGY
CRISPR is a technology which helps in modifying the sequence of DNA precisely, so that we can correct the mutation in the existing DNA sequence, which otherwise could lead to a disease.
1.1 CRISPR defence mechanism
CRISPR-Cas9 system is actually an adaptive bacterial immune system, which chooses RNA guided nucleases to cleave foreign genetic elements, but contemporarily, it is transforming bioscience by providing accurate genome editing and engineering tools.
CRISPR stands for Clustered Regularly Interspaced Palindromic Repeats, which was described in 1987, by Japanese researchers, as a series of short direct repeats interspaced with very short, unique sequences(spacers) in the genome of Escherichia coli.[1]
In 2005, it was discovered that many spacer sequences within CRISPRs, actually derive from viral origins, or it can be said that spacer sequences resemble bacteriophage DNAs.[1][2]
Later, CRISPR-Cas9 was proposed as an adaptive defence system in bacteria, which means that bacteria can acquire resistance against bacteriophages by integrating a genome fragment of the infectious bacteriophage into the CRISPR loci as spacers.
The dual tracrRNA: crRNA was then engineered as a single guide RNA (sgRNA) which retains two important characteristics-
That sgRNA 5’ end determines the DNA target site (by Watson Crick base pairing) whereas the 3’ side of the double stranded structure helps to bind to Cas9 protein.
Figure 1- Labelled diagram pointing the components of CRISPR Cas9 system [1]
Here PAM which is Protospacer Adjacent Motif, is a short sequence adjacent to the target site in invading viral DNA (or the required DNA that we want to cleave). It is critical for DNA binding.[1] So, the crux is that by creating a change in the guide sequence in the sgRNA, we can programme CRISPR Cas9 to target the required DNA sequence as long as it is adjacent to PAM.
This technology which can introduce double stranded breaks at defined positions can help to study genomic rearrangements and change the DNA sequences accordingly.
1.2 Applications
Because of the simplicity of the CRISPR/Cas technology, it has become a practical reality in the world of genome editing. Its introduction has been recorded as a milestone in medical science because it has the potential to treat a wide range of diseases including inherited genetic disorders such as cystic fibrosis, viral disorders such as HIV and cancer.
For example, already the CRISPR Cas system has been used to create a knockout of the CCR5 and C$BPB genes in human myeloid leukemia K562 cells.[2]
Also, this technology makes a promise to change the pace of agricultural research. Editing has been done in crop plants including rice, wheat, sorghum 1.4 and liverwort.[1]
1.3 Limitations
CRISPR Cas9 editing system introduces double strand breaks in DNA, which hampers precision.[3]
Another limitation is the inability to provide customized sequence just after cutting DNA at the target side.
Also, a lot of off-target editing is another demerit. It sometimes changes those genes which were not supposed to be altered. These shortcomings limit its use and pose safety risks in medicinal applications.
Recent introduction of prime editing in genome editing recovers the limitations posed by CRISPR-Cas9 system.
2. PRIME EDITING
Prime editing method permits the introduction of all twelve base to base conversions without inducing a DNA double stranded break. [3]
2.1 Advancements
2.1.1 The major improvement is the prime editing guide RNA (pegRNA) which programmes the Cas9 endonuclease. In this system, along with the spacer which is complementary to one DNA strand, a Primer Binding Site (PBS) region and an RNA template (for a new DNA sequence which will be introduced to the targeted gene after cutting) are also present. So, the PBS region is complementary to second strand, and it creates a primer for Reverse Transcriptase (RT), which is linked to Cas9(H840A) nickase. [3]
2.1.2 TheCas9 protein is heavily modified- it only nicks a single strand of the double helix instead of cutting both of them.[6] Another protein attached to Cas9 is a reverse transcriptase enzyme which will make a new DNA from the RNA template and insert it at the nicked target site. [4]
The RNA dependent DNA polymerase Reverse Transcriptase uses the pegRNA sequence as a template so that we can change the target sequence in a customized manner. Then both these strands are stabilised and integrated into the genome by the repair machinery present in cells.
Researchers take advantage of the repair system to eliminate the disease-causing genes and add customized DNA.[4]
Figure 2. Schematic Illustration of Prime Editing, as Proposed by Anzalone [5]
2.2 Benefits
Because prime editing does not cause a double stranded break, hence, it reduces the risks in genome editing. Also, the number of off-target effects observed for prime editors was less in comparison to CRISPR Cas editing system.[6]
Scientists are excited to use this technology and see rapid and innovative use in research community(spotlight)
Still a lot of evaluation needs to be done to adapt this technology for therapeutic applications.[5]
CONCLUSION
The introduction of CRISPR/Cas9 editing system has been revolutionary in the domain of gene editing. It is promising to take biogenetics to a much higher level by making the procedure of gene modification much easier. But it also poses some risks due to its limitations. Fortunately, a recent advancement has been seen by prime editors due to their capability of introducing single stranded breaks in DNA sequences. Though the whole methodology is still to be explored and edited to make it fit for practical use, but still its achievement is a marked milestone.
REFERENCES
Doudna J A; Charpentier E; The new frontier of genome engineering with CRISPR-Cas9; 2014
Pellagatti A; Dolatshad H; Valletta S, Boultwood J; Application of CRISPR/Cas9 genome editing to the study and treatment of disease; 2015
Marzec M; Zalewska AB; Hensel G; Prime Editing: A New Way for Genome Editing; 2020
Cohen J; Prime editing promises to be a cut above CRISPR, 2019 (pg 406)
Yan J; Cirincione A; Adamson B; Prime Editing: Precision Genome Editing by Reverse Transcription, 2019 (pg210-211)
Matsoukas I G; Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA; 2020
By Sameeksha
sameeksha8122001@gmail.com
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