Biology subjects

Terns, M. P.

Publications and source records attributed to Terns, M. P..

2 recordsLinked to original sources

bioRxiv2021

Structure and Function of an in vivo Assembled Type III-A CRISPR-Cas Complex Reveal Critical Roles of Dynamics in Activity Control

The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle, but its molecular basis for regulation remains unexplained. Recognition of the foreign RNA, or cognate target RNA (CTR), triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self-RNA, or noncognate target RNA (NTR) that differs from CTR only in its 3-protospacer flanking sequence. Here we captured four structures of in vivo assembled Lactococcus lactis Csm (LlCsm) by electron cryomicroscopy representing both the active and the inactive states. Surprisingly, in absence of bound RNA, LlCsm largely forms a minimal assembly lacking the Csm2 subunit with a stably bound catalytic subunit Csm1. Comparison of the minimal LlCsm structure and activities, both in vitro and in vivo, with those of fully assembled LlCsm reveals a molecular mechanism responsible for the viral RNA-activated and self RNA-inhibited activity of Csm1 through protein dynamics. Graphic Art Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/428455v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e9c428org.highwire.dtl.DTLVardef@4d3397org.highwire.dtl.DTLVardef@76184dorg.highwire.dtl.DTLVardef@1359dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Type III-A CRISPR systems as a versatile gene knockdown technology

CRISPR-Cas systems are functionally diverse prokaryotic anti-viral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis and S. thermophilus) were each expressed on a single plasmid in E. coli and the efficiency and specificity of gene knockdown was assessed by Northern blot analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and non-coding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.

microbiology↗