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AcrIIA5 v2 anti-CRISPR

Part Description

Anti CRISPR for Cas12g Anti CRISPR are small proteins (approximately, 12–193 amino acids) which have become, quickly a new method to regulate CRISPR system activity by altering the nuclease activity or hindering binding to the target gene.

Usage

We have thought to work on developing powerful protein designs to enable us to control the specificity and limit off-targeting of cas proteins. We have been relying on our protein evolution models that use deep learning to develop and predict new mutants of anti crispr proteins that are cas-specific and can act as an on-demand safety component for cas-based biosynthetic designs. This biosafety aspect is integrated with our therapeutic project for this year to develop our sensitive therapeutic design with natural brake elements (Acr-proteins) deployed to assure the safety of our design.

Structural Characterization

File:Dok1,png.png Figure 1 show docking score calculation for the anti-crispr proteins binding & affinity to cas12g

File:Dok2,png.png Figure 2 show docking score calculation for AcrIIA5V2 binding & affinity to cas12g

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the Acr||A5 v2 anti-crisper protein. The (R32E) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (G42K) had the least evolutionary fitness for Acr||A5 v2 anti-crisper protein As displayed in Figure(3).

Figure 3. shows the mutational landscape of the Acr A5 v2 anti-crisper protein.

Literarture Characterization

AcrIIA5 Inhibits Genome Editing Mediated by Type II-A and Type II-C CRISPR Systems in Mammalian Cells:
In this study, they temporarily co-transfected mouse Neuro-2a (N2a) or human HEK293T cells with plasmids encoding anti-CRISPR proteins, Cas9s, and their corresponding sgRNAs designed to target specific genomic regions in order to check whether AcrIIA5 might impede genome editing mediated by the four Cas9 homologs frequently utilised for genome-editing purposes in mammalian cells.The results demonstrate that AcrIIA5 effectively supresses four different Cas9 proteins (Nme1Cas9, SpyCas9, CjeCas9, and SauCas9) from editing the genome in vivo in both bacterial and mammalian cells. Additionally, AcrIIA5 blocks genome editing with an inhibitory strength comparable to that of earlier anti-CRISPRs as shown in figure 4.

Figure 4. AcrIIA5 Inhibits Genome Editing Mediated by Nme1Cas9, SpyCas9, CjeCas9, and SauCas9 in Mammalian Cells

References

1. Garcia, B., Lee, J., Edraki, A., Hidalgo-Reyes, Y., Erwood, S., Mir, A., ... & Davidson, A. R. (2019). Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used for genome editing. Cell reports, 29(7), 1739-1746.‏ Sequence and Features