A new experimental-bioinformatic method was developed for error and bias correction in high-throughput antibody sequencing.

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade− mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

BackgroundThe ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.ResultsThis study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.ConclusionsThese results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes.

Adaptive immunity is driven by the ability of lymphocytes to undergo V(D)J recombination and generate a highly diverse set of immune receptors (B cell receptors/secreted antibodies and T cell receptors) and their subsequent clonal selection and expansion upon molecular recognition of foreign antigens. These principles lead to remarkable, unique and dynamic immune receptor repertoires 1 . Deep sequencing provides increasing evidence for the presence of commonly shared (convergent) receptors across individual organisms within one species 2-4 . Convergent selection of specific receptors towards various antigens offers one explanation for these findings. For example, single cases of convergence have been reported in antibody repertoires of viral infection or allergy 5-8 . Recent studies demonstrate that convergent selection of sequence motifs within T cell receptor (TCR) repertoires can be identified on an even wider scale 9,10 . Here we report that there is extensive convergent selection in antibody repertoires of mice for a range of protein antigens and immunization conditions. We employed a deep learning approach utilizing variational autoencoders (VAEs) to model the underlying process of B cell receptor (BCR) recombination and assume that the data generation follows a Gaussian mixture model (GMM) in latent space. This provides both a latent embedding and cluster labels that group similar sequences, thus enabling the discovery of a multitude of convergent, antigen-associated sequence patterns. Using a linear, one-versus-all support vector machine (SVM), we confirm that the identified sequence patterns are predictive of antigenic exposure and outperform predictions based on the occurrence of public clones. Recombinant expression of both natural and in silico-generated antibodies possessing convergent patterns confirms their binding specificity to target antigens. Our work highlights to which extent convergence in antibody repertoires can occur and shows how deep learning can be applied for immunodiagnostics and antibody discovery and engineering.