Activation of recombinases at specific DNA loci by zinc-finger domain insertions.

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.

Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.

The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand. Here, we show that expression of promoter-associated short guide (sg)RNAs together with dead Cas9 (dCas9) fused to a Krüppel-associated box domains (KRABd) in combination with the transcription repression domain of methyl CpG-binding protein 2 (MeCP2) can lead to persistent gene silencing in mouse embryonic stem cells and in human embryonic kidney (HEK) 293 cells. Surprisingly, this effect is achievable and even enhanced in DNA (cytosine-5)-methyltransferase 3A and 3B (Dnmt3A-/-, Dnmt3b-/-) depleted cells. Our results suggest that dCas9-KRABd-MeCP2 fusions are useful for long-term epigenetic gene silencing with utility in cell biology and potentially in therapeutical settings.

Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases.

Protein intrinsically disordered regions (IDRs) play pivotal roles in molecular recognition and regulatory processes through structural disorder-to-order transitions. To understand and exploit the distinctive functional implications of IDRs and to unravel the underlying molecular mechanisms, structural disorder-to-function relationships need to be deciphered. The DNA site-specific recombinase system Cre/loxP represents an attractive model to investigate functional molecular mechanisms of IDRs. Cre contains a functionally dispensable disordered N-terminal tail, which becomes indispensable in the evolved Tre/loxLTR recombinase system. The difficulty to experimentally obtain structural information about this tail has so far precluded any mechanistic study on its involvement in DNA recombination. Here, we use in vitro and in silico evolution data, conformational dynamics, AI-based folding simulations, thermodynamic stability calculations, mutagenesis and DNA recombination assays to investigate how evolution and the dynamic behavior of this IDR may determine distinct functional properties. Our studies suggest that partial conformational order in the N-terminal tail of Tre recombinase and its packing to a conserved hydrophobic surface on the protein provide thermodynamic stability. Based on our results, we propose a link between protein stability and function, offering new plausible atom-detailed mechanistic insights into disorder-function relationships. Our work highlights the potential of N-terminal tails to be exploited for regulation of the activity of Cre-like tyrosine-type SSRs, which merits future investigations and could be of relevance in future rational engineering for their use in biotechnology and genomic medicine.

Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation.

Intracellular delivery of cargo molecules such as membrane-impermeable proteins or drugs is crucial for cell treatment in biological and medical applications. Recently, microfluidic mechanoporation techniques have enabled transfection of previously inaccessible cells. These techniques create transient pores in the cell membrane by shear-induced or constriction contact-based rapid cell deformation. However, cells deform and recover differently from a given extent of shear stress or compression and it is unclear how the underlying mechanical properties affect the delivery efficiency of molecules into cells. In this study, we identify cell elasticity as a key mechanical determinant of delivery efficiency leading to the development of "progressive mechanoporation" (PM), a novel mechanoporation method that improves delivery efficiency into cells of different elasticity. PM is based on a multistage cell deformation, through a combination of hydrodynamic forces that pre-deform cells followed by their contact-based compression inside a PDMS-based device controlled by a pressure-based microfluidic controller. PM allows processing of small sample volumes (about 20 µL) with high-throughput (>10 000 cells per s), while controlling both operating pressure and flow rate for a reliable and reproducible cell treatment. We find that uptake of molecules of different sizes is correlated with cell elasticity whereby delivery efficiency of small and big molecules is favoured in more compliant and stiffer cells, respectively. A possible explanation for this opposite trend is a different size, number and lifetime of opened pores. Our data demonstrates that PM reliably and reproducibly delivers impermeable cargo of the size of small molecule inhibitors such as 4 kDa FITC-dextran with >90% efficiency into cells of different mechanical properties without affecting their viability and proliferation rates. Importantly, also much larger cargos such as a >190 kDa Cas9 protein-sgRNA complex are efficiently delivered high-lighting the biological, biomedical and clinical applicability of our findings.

Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases.

The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-specific recombinases (SSRs) for genome editing applications. Previous work has revealed key residues for target site selectivity in the Cre/loxP and the related Dre/rox recombinase systems. However, enzymes in which these residues were changed to the respective counterpart only showed weak activity on the foreign target site. Here, we use molecular modeling and dynamics simulation techniques to comprehensively explore the mechanisms by which these residues determine target recognition in the context of their flanking regions in the protein-DNA interface, and we establish a structure-based rationale for the design of improved recombination activities. Our theoretical models reveal that nearest-neighbors to the specificity-determining residues are important players for enhancing SSR activity on the foreign target site. Based on the established rationale, we design new Cre variants with improved rox recombination activities, which we validate experimentally. Our work provides new insights into the target recognition mechanisms of Cre-like recombinases and represents an important step towards the rational design of SSRs for applied genome engineering.

MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting.

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

Systematic analysis of human protein complexes identifies chromosome segregation proteins.

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.