Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus

Abstract

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.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope

“…Given the reRNA-coding sequence and the TnpB-coding sequence are overlapped, truncation analysis of the reRNA element would also truncate the tnpB gene. To avoid such a scenario, a dual-plasmid system recently developed for this archaeon 54 was employed in which, one plasmid was used to express TnpB7 protein, and the other, for producing a guide RNA (reRNA) of variable sizes and for providing the repair template. As shown in Supplementary Fig.…”

“…Two plasmids, pSeSD 46 and pN1dAA 54 were used in the dual-plasmid gene editing system. To generate a host for gene editing with the dual plasmid system, we deleted the argD gene from the E233 parental strain using the endogenous type IA CRISPR system 21 .…”