<i>Staphylococcus aureus</i>Cas9 is a multiple-turnover enzyme

Yourik, Paul; Fuchs, Ryan T.; Mabuchi, Megumu; Curcuru, Jennifer L.; Robb, G. Brett

doi:10.1101/340042

Cited by 10 publications

(16 citation statements)

References 45 publications

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“…In all, as averaged across 5 targets, Spy Cas9 generated predominantly blunt-ended DNA target cleavage as reported previously 7 ( Figure 5B). Analysis of Sau Cas9 target cleavage produced almost entirely blunt-ended products as shown earlier 54 ( Figure 5B and S11 and Supplementary Table S5).…”

Section: Target Dna Cleavage By Cas9 Orthologssupporting

confidence: 75%

“…As shown in Figure 5A, these enzymes generated defined cut-sites, either blunt-ended or staggered as expected. This can be contrasted with Spy Cas9 target cleavage that, depending on target site, generated either blunt-end cuts, 1 nt 5' staggered termini due to RuvC post-cleavage trimming as observed previously 54 , or a mixture of both (Supplementary Figure S11). In all, as averaged across 5 targets, Spy Cas9 generated predominantly blunt-ended DNA target cleavage as reported previously 7 ( Figure 5B).…”

Section: Target Dna Cleavage By Cas9 Orthologsmentioning

confidence: 95%

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Biochemically diverse CRISPR-Cas9 orthologs

Gasiūnas

Young

Karvelis

et al. 2020

Preprint

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CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements.PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

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Section: Target Dna Cleavage By Cas9 Orthologssupporting

confidence: 75%

Section: Target Dna Cleavage By Cas9 Orthologsmentioning

confidence: 95%

Biochemically diverse CRISPR-Cas9 orthologs

Gasiūnas

Young

Karvelis

et al. 2020

Preprint

Self Cite

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“…SeqB lacked a 'NGG' protospacer adjacent motif (PAM) for the common S. pyogenes Cas9 (SpyCas9) for which DASH was originally described; therefore, we modified DASH to use S. aureus Cas9 (SauCas9) (Ran et al, 2015) . Moreover, in contrast to SpyCas9, SauCas9 is a multi-turnover enzyme (Yourik et al, 2019) , suggesting that SauCas9 would have higher cleavage efficiency, which was important since SeqB was present in multiple copies within a concatemer.…”

Section: Depletion Of Abundant Sequences By Hybridization (Dash) Of Smentioning

confidence: 99%

Single-cell profiling of Ebola virus infectionin vivoreveals viral and host transcriptional dynamics

Kotliar

Lin

Logue

et al. 2020

Preprint

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Ebola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo , we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5 ), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting. over-activation of innate and adaptive immunity underlies much of EVD pathology, rather than solely virus-mediated cytotoxicity (Geisbert et al., 2003a(Geisbert et al., , 2003b .

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“…We next tested the ability of the AcRanker-generated candidates to inhibit Staphylococcus aureus (SauCas9), another Cas9 commonly used for gene editing (50,51) to determine whether any of the candidates identified from self-targeting Streptococcus genomes had broader Cas9 inhibition activity. At a 25-fold excess relative to the SauCas9 RNP complex, ML3 and ML8 were able to inhibit SauCas9 dsDNA cleavage ( Figure 2C).…”

Section: Biochemical Validation Of Novel Acrs Identified By Acrankermentioning

confidence: 99%

Machine Learning Predicts New Anti-CRISPR Proteins

Eitzinger

Asif

Watters

et al. 2019

Preprint

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ABSTRACTThe increasing use of CRISPR-Cas9 in medicine, agriculture and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many such anti-CRISPRs have been found in mobile genetic elements that disable the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties that can be used for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method for identifying new potential anti-CRISPRs directly from proteomes using protein sequence information only. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking and extensively benchmarked it through non-redundant cross-validation and external validation. We then applied AcRanker to predict candidate anti-CRISPRs from self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA16 (ML1) and AcrIIA17 (ML8). We show that AcrIIA16 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA17 inhibits both SpyCas9 and SauCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

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Staphylococcus aureusCas9 is a multiple-turnover enzyme

Cited by 10 publications

References 45 publications

Biochemically diverse CRISPR-Cas9 orthologs

Biochemically diverse CRISPR-Cas9 orthologs

Single-cell profiling of Ebola virus infectionin vivoreveals viral and host transcriptional dynamics

Machine Learning Predicts New Anti-CRISPR Proteins

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