Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets.

Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off-target effects present major safety concerns for applications requiring high specificity. Approaches developed to date to mitigate this effect, including any of the increased-fidelity (i.e., high-fidelity) SpCas9 variants, only provide efficient editing on a relatively small fraction of targets without detectable off-targets. Upon addressing this problem, we reveal a rather unexpected cleavability ranking of target sequences, and a cleavage rule that governs the on-target and off-target cleavage of increased-fidelity SpCas9 variants but not that of SpCas9-NG or xCas9. According to this rule, for each target, an optimal variant with matching fidelity must be identified for efficient cleavage without detectable off-target effects. Based on this insight, we develop here an extended set of variants, the CRISPRecise set, with increased fidelity spanning across a wide range, with differences in fidelity small enough to comprise an optimal variant for each target, regardless of its cleavability ranking. We demonstrate efficient editing with maximum specificity even on those targets that have not been possible in previous studies.

Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs.

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9.

SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e.

Several advancements have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased-fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high-fidelity SpCas9 variant, free from the drawbacks of first-generation IFNs. In this study, we characterize the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells, comparing it to first-generation IFNs. SuperFi-Cas9 demonstrates strongly reduced activity but high fidelity features that are in many aspects similar to those of some first-generation variants, such as evo- and HeFSpCas9. SuperFi-cytosine (CBE3) and -adenine (ABE7.10) base editors, as well as SuperFi-prime editor show no meaningful activity. When combined with ABE8e, SuperFi-Cas9, similarly to HeFSpCas9, executes DNA editing with high activity as well as high specificity reducing both bystander and SpCas9-dependent off-target base editing.

Precision Global Determination of the B→X_{s}γ Decay Rate.

We perform the first global fit to inclusive B→X_{s}γ measurements using a model-independent treatment of the nonperturbative b-quark distribution function, with next-to-next-to-leading logarithmic resummation and O(α_{s}^{2}) fixed-order contributions. The normalization of the B→X_{s}γ decay rate, given by |C_{7}^{incl}V_{tb}V_{ts}^{*}|^{2}, is sensitive to physics beyond the standard model (SM). We determine |C_{7}^{incl}V_{tb}V_{ts}^{*}|=(14.77±0.51_{fit}±0.59_{theory}±0.08_{param})×10^{-3}, in good agreement with the SM prediction, and the b-quark mass m_{b}^{1S}=(4.750±0.027_{fit}±0.033_{theory}±0.003_{param}) GeV. Our results suggest that the uncertainties in the extracted B→X_{s}γ rate have been underestimated by up to a factor of 2, leaving more room for beyond-SM contributions.

Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5'G-extended sgRNAs.

Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach to mitigating its off-target effects. However, these variants are effective only in a restricted target space, and many of them are reported to work less efficiently when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low tolerance to 5'-extended, 21G-sgRNAs contributes, to a great extent, to their decreased performance. Here, we report the generation of Blackjack SpCas9 variant that shows increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations into previously reported increased fidelity variants make them effective with 21G-sgRNAs and increases their fidelity. Two "Blackjack" nucleases, eSpCas9-plus and SpCas9-HF1-plus are superior variants of eSpCas9 and SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA libraries with higher specificity and show similar activities whether delivered as plasmids or as pre-assembled ribonucleoproteins.

Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases.

The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

New Predictions for Λ_{b}→Λ_{c} Semileptonic Decays and Tests of Heavy Quark Symmetry.

The heavy quark effective theory makes model independent predictions for semileptonic Λ_{b}→Λ_{c} decays in terms of a small set of parameters. No subleading Isgur-Wise function occurs at order Λ_{QCD}/m_{c,b}, and only two subsubleading functions enter at order Λ_{QCD}^{2}/m_{c}^{2}. These features allow us to fit the form factors and decay rates calculated up to order Λ_{QCD}^{2}/m_{c}^{2} to LHCb data and lattice QCD calculations. We derive a significantly more precise standard model prediction for the ratio B(Λ_{b}→Λ_{c}τν[over ¯])/B(Λ_{b}→Λ_{c}µν[over ¯]) than prior results, and find the expansion in Λ_{QCD}/m_{c} well behaved, addressing a long-standing question. Our results allow more precise and reliable calculations of Λ_{b}→Λ_{c}ℓν[over ¯] rates, and are systematically improvable with better data on the µ (or e) modes.

Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.

Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.

BACKGROUND: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. RESULTS: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s. CONCLUSIONS: No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.

Towards More Precise Determinations of the Quark Mixing Phase ß.

We derive a new flavor symmetry relation for the determination of the weak phase ß=ϕ_{1} from time-dependent CP asymmetries and B→J/ψP decay rates. In this relation, the contributions to sin2ß proportional to V_{ub} are parametrically suppressed compared to the contributions in the B→J/ψK^{0} time-dependent CP asymmetry alone. This relation uses only SU(3) flavor symmetry, and does not require further diagrammatic assumptions. The current data either fluctuate at the 2σ level from expectations, or may hint at effects of unexpected magnitude from contributions proportional to V_{ub} or from isospin breaking.

Implications of the dimuon CP asymmetry in B(d,s) decays.

The D0 Collaboration reported a 3.2σ deviation from the standard model (SM) prediction in the like-sign dimuon asymmetry. Assuming that new physics contributes only to B(d,s) mixing, we show that the data can be analyzed without using the theoretical calculation of ΔΓ(s), allowing for robust interpretations. We find that this framework gives a good fit to all measurements, including the recent CDF Collaboration S(ψϕ) result. The data allow universal new physics with similar contributions relative to the SM in the B(d) and B(s) systems, but favors a larger deviation in B(s) than in B(d) mixing. The general minimal flavor violation framework with flavor diagonal CP violating phases can account for the former case and remarkably even for the latter case. This observation makes it simpler to speculate about which extensions with general flavor structure may also fit the data.

Implications of the measurement of the Bs(0)Bs(0) mass difference.

We analyze the significant new model independent constraints on extensions of the standard model (SM) that follow from the recent measurements of the Bs(0)Bs(0) mass difference. The time-dependent CP asymmetry in Bs-->psiphi, S(psiphi), will be measured with good precision in the first year of CERN Large Hadron Collider (LHC) data taking, which will further constrain the parameter space of many extensions of the SM, in particular, next-to-minimal flavor violation. The CP asymmetry in semileptonic Bs decay, ASL(s), is also important to constrain these frameworks, and could give further clues to our understanding the flavor sector in the LHC era. We point out a strong correlation between S(psiphi) and ASL(s) in a very broad class of new physics models.