Off-targets in CRISPR and Ways to reduce them
Off-targets in CRISPR: CRISPR is genetic scissors which are used to cut targeted sequences in the genome. Off-targets sites are those sites which are not targeted/Non-interested sites where Cas9 binds. This causes cleavage in unintended genomic sites the effects of this “Unintended cleavage” is called as off-target effects.
If the CRISPR enzyme cleaves the DNA at random sites it causes concerns about the uses and the application of this technique.
How do we reduce Off-targets?
So, here are a few ways in which one can reduce the Off-targets and increase the efficiency of CRISPR editing
- Re-engineering the Guide RNA — gRNA
- Employing different Cas9 proteins
- Adapting Non-Viral delivery methods
- Using Base editors
- Regulating temperatures
- Using Anti-CRISPR Proteins
- Re-engineering the Guide RNA — GRNA
Guide RNA helps to identify the cleavage site in the DNA. So, designing a proper gRNA is an essential way to reduce Off-targets.
- 40% — 60% of GC contents in the gRNA increase the on-target performance.
- Guanine at 20th position and Cytosine at 16th position also increases the ontarget editing.
- sgRNA (Single guide RNA) of length 18 base pair can reduce the mutations at Off-target sites.
2. Employing different Cas9 proteins
Cas9 protein is an RNA-guided endonuclease which is derived from wild type Streptococcus pyogenes; this gRNA seems to produce high off-target effects. Different Cas9 proteins can reduce this to an extent.
Some Cas9 variants are
- SaCas9 (Staphylococcus aureus) — Has much higher on-target mutagenesis.
- StCas9 (Streptococcus thermophilus) — More specific.
- CjCas9 (Campylobacter jejuni) — Reduced off-target activity.
3. Different delivery methods
Methods used to introduce the gRNA and Cas9 plays a vital role in the accuracy of the targets.
Recently Non-viral CRISPR delivery methods have been employed. Some of these methods are
- Electroporation — Delivering the gRNA and Cas9 as RNP (Ribonucleoprotein) complex is better than plasmid transfection
- Using biomaterials Cell-derived nanovesicles, Magnetic nanoparticles (MNP) and Polyethyleneimine methods have shown to be nontoxic and have low off target effects. MNPs is considered to be the best delivery method for CRISPR/Cas9.
4. Using Base editors
Base editing is a gene-editing approach that uses the CRISPR/Cas9 system and other enzymes to cause direct point mutations without making double-stranded DNA breaks (DSBs).
Unintended mutation (Off-targets) occur usually from double-stranded DNA breaks (DSBs) followed by Non-homologous end joining (NHEJ). So using base editors can reduce this effect.
- Cytosine Base Editor = mediate a C to T change (or a G to A change on the opposite strand)
- Adenine Base Editor = mediate an A to G change (or a T to C change on the opposite strand)
5. Regulating temperatures
The temperature of the growth chamber influences on & off-target mutations.
- 37°C is an optimal temperature, it affects the Cas9 cleavage and increases the chances of on-target mutations.
6. Using Anti-CRISPR Proteins
Anti-CRISPR proteins are produced by phages to evade the CRISPR system.
- Recently, it has been found that using Anti-CRISPR proteins after a certain time (6 hours) can reduce off-target effects.
- AcrIIA4, an inhibitor protein from the Listeria bacteriophage reduced by fourfold the off-target effects of a CRISPR-Cas9
REFERENCE:
- Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020). Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells, 9(7), 1608
- Hajiahmadi, Z., Movahedi, A., Wei, H., Li, D., Orooji, Y., Ruan, H., & Zhuge, Q. (2019). Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants. International journal of molecular sciences, 20(15), 3719. https://doi.org/10.3390/ijms20153719
- Han, H. A., Pang, J. K. S., & Soh, B. S. (2020). Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing. Journal of Molecular Medicine, 1–18.
- https://news.berkeley.edu/2017/07/12/anti-crispr-proteins-decrease-off-target-side-effects-of-crispr-cas9/
- https://www.the-scientist.com/news-opinion/anti-crispr-protein-reduces-off-target-effects-31255
