No abstract

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and Cterminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.Protein splicing is a novel mode of gene expression that has been described in mesophilic bacteria and yeast and in extremely thermophilic archaea (1-6). It is a process in which a single gene directs the synthesis of two separate proteins by the precise excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and C-terminal regions, the exteins, to yield two new proteins (7). Some of the excised inteins are homing endonucleases that catalyze lateral transfer of their DNA coding sequences by an intein homing mechanism (8 -11), whereas the ligated exteins are usually enzymes with a specific cellular function.Efficient protein splicing also occurs when inteins are transferred into heterologous proteins, suggesting that all structural and catalytic elements needed for the splicing reaction reside in the inteins plus the first C-extein residue (4,(12)(13)(14). The protein splicing function of inteins is independent of their homing endonuclease activity (15) and depends on highly conserved amino acid residues at both splice junctions. A hydroxyl-or thiol-containing residue (Ser, Thr, or Cys) is always present at the positions that immediately follow the two splice junctions, and the sequence His-Asn is invariant at the intein C terminus. Substitution of any of these conserved residues retards or abolishes protein splicing (2,12,13,15).The fact that the thermophilic archaeal inteins in a foreign protein context undergo efficient splicing only at elevated temperatures (25-65°C) opened the way for the development of an in vitro system to study the mechanism of protein splicing (14). We constructed a fusion protein containing the intein from the thermostable DNA polymerase of Pyroccocus sp. GB-D, which could be expressed i...