Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.

The evolution of CRISPR-Cas systems and their possible origin from mobile genetic elements of transposons are currently being actively studied. Recently, unique systems have been discovered in a new group of Asgardarchaea that presumably function as transposons and contain Cas1-like proteins. In this study, the genetic and biochemical technologies were used, along with electrophoresis, affinity chromatography, and high-resolution mass spectrometry, to obtain and partially characterize a recombinant version of one of these proteins, Cas1_3. In particular, it was shown to have the ATPase activity, the quantitative value of the latter being determined by the spectrophotometric method. The results obtained may be useful in understanding the mechanisms of functioning the potential ancestor of CRISPR-Cas systems.

The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

ABSTRACT CRISPR-Cas12d is a distinct V-D type system discovered in the metagenomes of Candidate Phyla Radiation bacteria. It stands out from most closely related systems due to its 17-19 nucleotide short spacer region and specialized stabilizing scoutRNAs. We made significant improvements to this system by modifying its scoutRNA to create sgRNA, which greatly simplifies its use. We found mutations in the RuvC domain of the effector protein KbCas12d that resulted in loss of nuclease activity. We obtained two catalytically inactive dKbCas12d variants: D827A and E913A. Using the optical tweezers technique, we demonstrated the high specificity of dKbCas12d in binding targets on individual DNA molecules. Engineered sgRNA and catalytically inactive dKbCas12d variants have promising applications in biotechnology for the precise regulation of gene expression and molecular diagnostics.

Type II CRISPR-Cas9 RNA-guided nucleases are commonly used for genome engineering. To date, all characterized Cas9-based genome editors, including the widely used SpCas9, have limitations such as their relatively large size and restriction of targets flanked by a specific PAM sequence. Here, we biochemically characterized more compact SpCas9 ortholog, SuCas9, from Streptococcus uberis , a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5′-NNAAA-3′ PAM, efficiently cleaves DNA in vitro , and is active in human cells. The study of SuCas9 has the potential to expand the range of applications of CRISPR-Cas9 enzymes in medicine and biotechnology. ### Competing Interest Statement The authors have declared no competing interest.

AbstractType II CRISPR-Cas9 RNA-guided nucleases are commonly used for genome engineering. To date, all characterized Cas9-based genome editors, including the widely used SpCas9, have limitations such as their relatively large size and restriction of targets flanked by a specific PAM sequence. Here, we biochemically characterized more compact SpCas9 ortholog, SuCas9, fromStreptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5′-NNAAA-3′ PAM, efficiently cleaves DNAin vitro, and is active in human cells. The study of SuCas9 has the potential to expand the range of applications of CRISPR-Cas9 enzymes in medicine and biotechnology.

CRISPR (clustered regularly interspaced short palindromic repeats) Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Understanding features of the HIV-1 transmission process has the potential to inform biological interventions for prevention. We have examined the transmitted virus in a cohort of people who inject drugs and who are at risk of HIV-1 infection through blood contamination when injecting in a group. This study focused on seven newly infected participants in St. Petersburg, Russia, who were in acute or early infection. We used end-point dilution polymerase chain reaction to amplify single viral genomes to assess the complexity of the transmitted virus. We also used deep sequencing to further assess the complexity of the virus. We interpret the results as indicating that a single viral variant was transmitted in each case, consistent with a model where the exposure to virus during transmission was limited. We also looked at phenotypic properties of the viral Env protein in isolates from acute and chronic infection. Although differences were noted, there was no consistent pattern that distinguished the transmitted variants. Similarly, despite the reduced genetic heterogeneity of the more recent subtype A HIV-1 epidemic in St. Petersburg, we did not see reduced variance in the neutralization properties compared to isolates from the more mature subtype C HIV-1 epidemic. Finally, in looking at members of injecting groups related to the acute HIV-1 infection/early subjects, we found examples of sequence linkage consistent with ongoing and rapid spread of HIV-1 in these groups. These studies emphasize the dynamic nature of this epidemic and reinforce the idea that improved prevention methods are needed.

Despite Synechococcus picocyanobacteria being important in lacustrine ecosystems, they have received less attention than their marine counterparts. We describe for the first time, using 454 pyrosequencing, the diversity of Lake Onego cyanobacteria. The majority (97.52%) of 16S rRNA gene sequences observed in Synechococcus collected from the epilimnion belonged to OTUs (operational taxonomic units) I-XVI. The obtained pyrotags were classified, together with specially created database of 770 reference sequences, via the M-pick algorithm and compared with traditional (cited in the literature) assemblages, which for reason of reliability were reappraised. The entire set of sequences were attributed to freshwater Synechococcus phylotypes (FSP) 1-45. Most of the Lake Onego Synechococcus sequences (47.47%) belonged to OTU VII within FSP 14, which comprised ubiquitous strains, whereas the low-abundance FSP 20 (5% of sequences) demonstrated a limited geographical distribution. The study of connection between Lake Onego phylotypes and those in other large lakes should be continued because no analysis of a similar scale has been undertaken as yet. Cluster analysis via weighted UniFrac revealed no correlation between the spatial distribution of the lake's Synechococcus community and limnological parameters. Only two Synechococcus strains (both phycoerythrin-lacking) were obtained in culture confirming that lacustrine picocyanobacteria are difficult to culture.

Mitochondrial gene NADH dehydrogenase subunit 1 (nad1), β-tubulin gene, and elongation factor 1-alpha (tef) were used to characterize and to identify 42 Lecanicillum spp. isolates (former complex species Verticillium lecanii Zimm. Viegas) and to study the phylogenetic relationships in this group. Within the isolates under investigation, Lecanicillium muscarium was the most common species (about 70% of all isolates, collected on the different hosts, predominantly on the insects from the order Hemiptera). Based on nad1 sequencing four main molecular haplotypes were revealed. All four haplotypes have Holarctic origin. Most of them were isolated in the Central part of Russia. One haplotype showed a specific association with the certain geographical area, limited to southwest Georgia and the Krasnodar Territory. For most strains their affiliation to species L. muscarium, L. longisporum, L. psalliotae, L. pissodes were confirmed by the phylogenetic tree, based on the combined sequences of nad1, β-tub, and tef genes. Only five strains of haplotype C and strain F-2643 could not be identified to any present Lecanicillium species and their position remains ambiguous. Thus, the use of multilocus molecular approach based on these genes was useful to identify the Lecanicillium species. Inter-simple sequence repeat (ISSR) study evaluated a high diversity among the L. muscarium strains. The topology of the NJ-tree based on the ISSR-PCR markers has shown the genetic relationships with the support values 62-91% between L. muscarium isolates.

Rare bacterial biosphere (RBB) is a large and probably predominant sector of bacterial diversity, which is specifically represented by small populations. Although some RBB components have been characterized phenotypically (actualistic objects), it has been mainly described as a set of virtual objects, i.e., of the 16S rRNA gene sequences from environmental DNA samples, which are grouped into phylotypes (operational taxonomic units, OTUs). The upper OTU threshold for RBB is presently not standardized. It is usually ~1% of the sum of OTU sequences in the metagenome library, or five sequences per OTU in absolute values. The analyzed RBB objects include (1) virtual and actualistic objects; (2) autochthonous and allochthonous forms; (3) vegetative and differentiated cells; (4) dead bacteria and free DNA; and (5) artifacts and informational gaps. The RBB phenomenon has not been sufficiently explained. According to some concepts, the RBB objects are rare due to restrictive action of unfavorable environmental factors. According to others, they utilize a successful adaptive strategy of low abundance, which facilitates higher genetic diversity, dispersal and colonization of new niches, and microbial conversion of specific substrates. Since RBB was revealed only in the early 2000s and is still poorly studied, its role in organic evolution and its place in the ecosystems should be determined by future research. The information on the RBB composition, distribution, and functions will be important for bacteriology, while some cultured species may be of basic or applied importance.

We report the first insight, using 454 pyrosequencing, into the diversity of bacterial plankton from Lake Lagoda (Russia), one of the world's largest lakes and the largest European lake. The majority of 16S rRNA sequences recovered from the lake epilimnion belonged to the phyla Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia and Cyanobacteria. Proteobacterial pyrotags were mainly from the classes Betaproteobacteria and Alphaproteobacteria. These data, supplemented with clone assignment to the freshwater bacterial clades, demonstrate that the diversity of Lake Ladoga bacterial plankton is similar to other large freshwater lakes. At the same time, the cyanobacterial sequence abundance was low, with 2.5% of the universal primer-based amplicons derived from DNA templates sampled at three hydrographic stations. Similar to the communities in other large lakes, 85% of group-specific primer based sequences corresponded to Synechococcus. Most of the abundant (n>5 per library) Synechococcus sequences comprising operational taxonomic units (OTUs) I − XVIII belonged to the widely distributed clusters MH305 and LB03 (47% and 12.6% of sequences, respectively). OTUs I–IV (19%) comprised a putative assemblage termed Group LL. A small proportion of Synechococcus sequences (~4%), with n≤5 per library, belonged to low-abundance OTUs 1′42′. Five phycoerythrin-lacking Synechococcus strains were cultured; four of them clustered with Synechococcus Group A, whereas the fifth strain which did not match any pyrotag clustered with Synechococcus Group I. Group LL representatives were not cultured successfully. In conclusion, Lake Ladoga bacterial plankton shares common features with that in other (large) lakes and has obtained some specificity probably under low temperature selection.