RESUMO
The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.
Assuntos
Pareamento Incorreto de Bases , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Humanos , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/química , DNA/química , DNA/metabolismo , Simulação de Dinâmica Molecular , RNA/química , RNA/metabolismo , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/química , Células HEK293 , Edição de GenesRESUMO
High-altitude mammals are often subject to specific environmental obstacles, which exert selective pressure on their physiological and morphological traits, hence driving their evolutionary processes. It is anticipated that these circumstances will lead to the adaptive evolution of protein-coding genes (PCGs) in the mitochondrial genome, which play a crucial role in the oxidative phosphorylation system. In this study, we have generated the complete mitochondrial genome of the Badri breed of Bos indicus inhabiting a high-altitude environment to test the signatures of adaptive evolution on PCGs and their phylogenetic relationships. The complete mitogenome of the Badri breed is 16,339 bp and most tRNAs showed typical clover-leaf secondary structure with a few exceptions, like trnS1 and trnS2 without DHU arm and trnK without DHU loop. Comparative analysis of PCGs indicated that cox1 is the most conserved, while atp6 is the most variable gene. Moreover, the ratios of non-synonymous to synonymous substitution rates indicated the purifying selection (Ka/Ks < 1) in the protein-coding genes that shape the diversity in mitogenome of Bos indicus. Furthermore, Branch-site model (BSM) suggested that cox1, cox2, nad3, nad4L, and nad6 underwent stronger purifying selection (ω < 1) than other PCGs in 15 breeds of 4 species, including Badri. BSM also detected 10 positive sites in PCGs and one in 13 PCGs concatenated dataset. Additional analyses in Datamonkey indicated 11 positive sites and 23 purifying sites in the concatenated dataset, a relaxation of selection strength in nad3, and no evidence of episodic diversifying selection in any PCGs. Phylogeny revealed the sister relationship of the Badri with other breeds of Bos indicus as well as Bos frontalis (Gayal-2). The mitogenome of the Badri breed is an important genomic resource for conservation genetics of this species and also contributes to the understanding of the adaptive evolution of mitochondrial protein coding genes.