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  • Rational design of unrestri...
    Zhao, Pengpeng; Bi, Xiaonan; Wang, Xiaoning; Feng, Xu; Shen, Yulong; Yuan, Guanhua; She, Qunxin

    mLife, March 2024, Letnik: 3, Številka: 1
    Journal Article

    Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1‐based vectors consistently failed to yield any stable host–vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1‐based and pRN2‐based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model. Impact statement When pRN1 was employed for vector construction in Saccharolobus islandicus REY15A, pRN1‐derived vectors were not stable in this archaeon. Here, we show that pRN1 orf904 encoding a putative replicase on pRN1 carries a DNA segment to be targeted by the host I‐A CRISPR system. By designing mutated target sequences that evade the CRISPR immunity, efficient plasmid vectors were obtained with mutated pRN1 backbones. This strategy could be applied in developing host–vector systems for other microorganisms with plasmids or viruses carrying CRISPR target sequences. Moreover, the resulting dual vector system would facilitate genetic studies with this important crenarchaeal model.