Inside our cells, we carry the genetic material (DNA) inherited from our parents which act as the machine to produce the proteins required for the proper functioning of our body. Just like humans, viruses and bacteria also have DNA or RNA as their genetic material. A group of researchers observed that bacteria contain certain repeating segments separated by short non-repeating segments(Spacer) in their genetic material, which later was named as “clustered regularly interspaced palindromic repeat (CRISPR)“, helped bacteria in attaining immunity against viruses with the help of a DNA cleaving enzyme(Cas9).
How bacteria use CRISPR to provide immunity against invading virus?
Whenever there is an entry of virus or bacteriophage inside a bacteria a part of the virus genetic material known as protospacer which lies close to the PAM (protospacer adjacent motifs) sequence is cleaved and integrated into the spacer region of the bacterial genome. Later the CRISPR-RNA (guide RNA) is transcribed from the CRISPR region, which guides CAS9 nuclease enzyme to detect and destroy (cleave) DNA of a virus, which is complementary to the guide RNA, whenever there is an entry of a similar virus attack.
Soon researchers start using CRISPR-Cas9 as a gene-editing tool for various diseases. Since virus infection in eukaryotes(plants, human) is posing a serious threat to the survival of the being, researchers tried to manipulate the CRISPR-Cas9 system and used it for protecting eukaryotes against viruses.
Use of CRISPR-Cas9 in plants
In 2015 a research article was published which studied the CRISPR-Cas9-mediated viral interference in plants. .A N. benthamiana plant was used as a host, by overexpressing CAS9 proteins. It was Further, infected by a tobacco rattle virus (TRV) vector that was cloned with three guide RNAs(IR sgRNA, CP sgRNA, Rep ORF sgRNA) attacking different regions of a tomato yellow leaf curl virus (TYLCV).
Flowchart representation of the experimental design.
Comparing sgRNAs targeting the CP, the RCRII motif of Rep, and the IR sequences
Though all the sgRNAs were capable of mediating targeted cleavage of the TYLCV genome only sg RNA against IR was able to reduce viral replication and decrease symptoms. The possible reason behind these differences may be that the CP, the RCRII sequences codes for proteins which are less required in the completion of the viral cycle.
- Co-delivery of multiple sgRNAs via the TRV system
When separate RNA2 genomes were used there was no additive effect observed and the reason behind it might be the way TRV vector infection occurs as the different sgRNAs are not made in the same cell so unable to simultaneously cleave the TYLCV. On the other hand, construction of a TRV RNA2 genome containing multiple sgRNAs led to multiplexed editing in single cells, resulting in an additive effect.
Attenuation of replication of TYLCV
The guide RNA attacking the intergenic region of the tomato yellow leaf curl virus (TYLCV) lead to attenuation of the replication of the virus.
An investigation was carried out whether the attenuated replication of TYLCV was due to targeted cleavage or modification of the genome, rather than simply to interference with the replication machinery resulting from binding by the CRISPR/Cas9 complex.
- Restriction site loss
Every genetic material DNA or RNA has a certain sequence, which is recognized by restriction enzymes and is cleaved at the site of recognition. The TYLCV has an intergenic region of
20-nucleotide.The target sequence of the IR of the TYLCV contains a recognition sequence for SspI endonuclease but when there is a modification by CRISPR-Cas9 the recognition sequence is eliminated and thus replication attenuation occurs.
- Use of T7 endonuclease 1(T7E1)
Whenever there is a double-strand break a non-homologous end joining occurs which results in the formation of new recombination leading to the formation of mismatched base pairs and thus DNA distortion occurs. T7E1 is an enzyme, which detects such structural deformities in double-stranded DNA and cleaves it. Thus, cleavage by cas9 nuclease led to such deformities and which is recognized and cleaved by T7E1 thereby confirming that CRISPR-Cas9 mediated modification is leading to replication attenuation.
Targeting multiple DNA viruses using a single sgRNA
A certain sequence in the genetic material of the viruses are conserved and the same sequence is present in multiple viruses. When guide RNA is designed against such conserved similar sequences present in the viral genome, it can be used against multiple viruses. For example, A certain sequence of the origin of replication in the intergenic region is conserved in all geminiviruses. When IR-sgRNA is designed that contains the invariant TAATATTAC sequence common to all geminiviruses, it can be used for modification of DNA of the entire family
Generation of virus variants with better adaptation
Viruses becoming resistant to antibiotics or any technique like CRISPR-Cas9 is a serious growing threat in today’s world. When there is a selection pressure on the virus for their survival lead to the generation of variants with better adaptation. Alteration or mutation in the sequences like IR and PAM makes CRISPR-Cas9 nonfunctional to that particular viral strain.
To maintain the replication of a new viral variant, the key replication enzymes that bind to this sequencea also mutate to recognize the new sequence. This issue can be tackled by
- Targeting two viral sequences for cleavage which will lead to the destruction of the viral genome, reducing the likelihood of DNA repair and generation of CRISPR-Cas9 infectious viral variants.
- Designing of sgRNA molecules specific for the new variant sequences.
After evaluating the results of the feasibility of using CRISPR-Cas9-mediated immunization against DNA viruses in plants the researchers concluded that CRISPR-Cas9 system can be used for targeted cleavage of the TYLCV genome. As targeting the TYLCV IR led to a significant reduction in TYLCV accumulation and disease symptoms. The researchers gave forward certain aspects of this gene editing tool:
1) The ability to target multiple DNA viruses simultaneously using a single sgRNA targeting a conserved sequence
2) The potential to overcome resistance by targeting newly evolved viral strains with new sgRNAs
3) Extending the utility of the CRISPR-Cas9 system for viral interference in plants will create a platform for understanding natural resistance and immune functions. At the same time, it will provide biotechnologists with a powerful tool for producing crop plants resistant to multiple viral infections.
“ CRISPR-Cas9 is changing the modern era of gene editing creating new possibilities for engineering plants resistant to DNA viruses.“
Ali, Z., Abulfaraj, A., Idris, A., Ali, S., Tashkandi, M. and Mahfouz, M.M., 2015. CRISPR/Cas9-mediated viral interference in plants. Genome biology, 16(1), p.238.