Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent non-homologous end-joining and polymerase theta (POLQ)-mediated end-joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect homology-directed repair of the inactivated paromomycin resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate homology-directed repair/gene inactivation. Our data suggests that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced homology-directed repair and random integration of transgenes, while KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in Chlamydomonas reinhardtii and can be used for future development of algal biotechnology.

Gross Chromosomal Rearrangements in Kluyveromyces marxianus Revealed by Illumina and Oxford Nanopore Sequencing

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research.

Gross Chromosomal Rearrangements in Kluyveromyces Marxianus Revealed by Illumina and Oxford Nanopore Sequencing

Kluyveromyces marxianus (K. marxianus) is a newly emerging industrially relevant yeast. It is known to possess a highly efficient Non-Homologous End Joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the Gross Chromosomal Rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in Eukaryotes, which could potentially provide insights for cancer research.

Principles of Genetic Engineering

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Development of an amenable system for site-specific addition to a maize chromosome

Currently, transgenic maize is produced by random integration of a transgenes into the plant. This works for single genes, but not as well for multiple traits. Identifying plants that contain several transgenes becomes a very difficult task. Gene stacking at a single location in the genome would make combining multiple transgenes into plants a simpler process. This project focused on the development of a system would allow for transgenes to be sequentially added to a specific site in the maize genome. The system utilizes two recombinases, Cre recombinase and _C31 Integrase, to remove a selectable marker and to integrate transgenes. An initial construct containing a selectable marker, flanked by LoxP sites, which are acted upon by Cre recombinase, and an attP site, were transformed. The selectable marker was then removed from the integrated transgene by exposure to Cre recombinase. Two amendment constructs enable modification of the integrated construct by utilizing complementary attP and attB sites, which are acted upon by _C31 Integrase. The amendment constructs contain cargo and a promoterless selectable marker which, upon successful recombination with the target site, restores expression of the selectable marker. Successful demonstration of this system simplifies generation of multi-transgene plants, and the assembly of multi-gene pathways in plants.

Development of a Transformation Method for Metschnikowia borealis and other CUG-Serine Yeasts

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowia borealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications.