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.

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.

Expression of the Prion Protein Family Member Shadoo Causes Drug Hypersensitivity That Is Diminished by the Coexpression of the Wild Type Prion Protein.

The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease.

Preparation of semisynthetic lipoproteins with fluorescent cholesterol anchor and their introduction to the cell membrane with minimal disruption of the membrane.

The exogenous introduction of fluorescent lipoproteins into cell membranes is a method for visualizing the cellular traffic of membrane associated proteins, and also for altering the cell surface in a controlled manner. In order to achieve the cell membrane anchoring of proteins and their subsequent fluorescence based detection, a cholesterol derivative was designed. The headgroup of the novel cholesterol anchor contains a fluorescent reporter and a thiol reactive maleimide for protein conjugation. Protein conjugation was demonstrated by the addition of a green fluorescent maleimido anchor to the C-terminus of a Cys extended red fluorescent protein, mCherry. The resulting dual fluorescent cholesteryl lipoprotein was successfully separated from the micellar associates of the surplus fluorescent lipid anchor without denaturing the protein, and the lipoprotein containing only the covalently linked, stoichiometric fluorescent lipid was efficiently delivered to the plasma membrane of live cells. It was demonstrated that the membrane fluorescence could be directly assigned to the protein-anchor conjugate, because no excess of fluorescent lipid species were present during the imaging experiment and the protein and anchor fluorescence colocalized in the cell membrane. Molecular dynamics simulations and subsequent trajectory analysis suggest also the spontaneous and stable membrane association of the cholesterol anchor. Thus, the method could be beneficially applied for studying membrane associated proteins and for preparing mimetics of glycosylphosphatidylinositol (GPI)-anchored proteins to target cholesterol-rich membrane microdomains.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein's physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein-protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

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.

Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine.

Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a ß-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants.

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.

The prion protein family member Shadoo induces spontaneous ionic currents in cultured cells.

Some mutant forms of the cellular prion protein (PrPC) carrying artificial deletions or point mutations associated with familial human prion diseases are capable of inducing spontaneous ionic currents across the cell membrane, conferring hypersensitivity to certain antibiotics to a wide range of cultured cells and primary cerebellar granular neurons (CGNs). These effects are abrogated when the wild type (WT) form is co-expressed, suggesting that they might be related to a physiological activity of PrPC. Interestingly, the prion protein family member Shadoo (Sho) makes cells hypersensitive to the same antibiotics as mutant PrP-s, an effect that is diminished by the co-expression of WT-PrP. Here, we report that Sho engages in another mutant PrP-like activity: it spontaneously induces large ionic currents in cultured SH-SY5Y cells, as detected by whole-cell patch clamping. These currents are also decreased by the co-expression of WT-PrP. Furthermore, deletion of the N-terminal (RXXX)8 motif of Sho, mutation of the eight arginine residues of this motif to glutamines, or replacement of the hydrophobic domain by that of PrP, also diminish Sho-induced ionic currents. Our results suggest that the channel activity that is also characteristic to some pathogenic PrP mutants may be linked to a physiological function of Sho.

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity.

The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.