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Stanford Researchers Develop Multiplexed, Precision Genome Editing Tool for Yeast


NEW YORK (GenomeWeb) – A group led by Stanford University researchers has created a new multiplex precision genome editing tool for studying the genetic basis of phenotypes in Saccharomyces cerevisiae.

Justin Smith, first author and postdoctoral fellow at Stanford, noted that his team chose the yeast species as a target because of its efficient homologous recombination ability, in addition to the research group's deep history of performing quantitative trait locus studies on yeast.

"We're aiming to refine the previous technology in order to map individual variants in the species," he explained.

In a three-part study published earlier this month in Nature Biotechnology, Smith and his colleagues developed a CRISPR-Cas9 method in S. cerevisiae for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) that overcomes important shortcomings in current approaches. The team's editing tool uses array-synthesized guide-donor oligonucleotides for plasmid-based high-throughput editing and includes genomic barcode integration to prevent plasmid barcode loss and to enable stable phenotyping.

The team began by introducing durable, genome-integrated barcodes as guides instead of plasmid barcodes in order to target both the plasmid and a chromosomal barcode locus. The barcodes attach to the guide-donor pairs, which successfully integrate themselves into the yeast genomes and create a signal to reveal their locations.

By using genome-integrated barcodes, the researchers therefore removed the need for plasmid maintenance, allowing marker-free variant tracking and one-to-one agreement of barcode counts to strain abundance.

"The problem with plasmid-based barcodes is that plasmids can often get lost and you usually need to grow them in a special media, either in the presence of a drug or an auxotroph to maintain the barcode," Smith explained. "With our system, it's genome integrated, and once it's done, those strains are always barcoded. We don't have to worry about maintaining plasmid or plasmid copy number."

The team designed the guide-donor plasmid to introduce a premature termination codon into the ADE2 open reading frame within the yeast genome. By disrupting ADE2, the researchers caused yeast samples to turn red, allowing the group to directly identify edited colonies. The team tested three different Pol III promoters — RPR1, SNr52, and tRNA-Tyr(SUP4)- HDV) — to drive expression of the guide pair and found similar kinetics of barcode integration upon Cas9 induction. By the 11th generation, PCR amplification showed that almost the entire yeast cell population had integrated the donor DNA into its genome. Overall, the researchers found that precision editing, genomic barcode integration, as well as guide-donor plasmid self-destruction all reached completion by the 11th generation.

The researchers also found that they could increase homologous recombination efficiency more than fivefold by actively recruiting a tandem array of four LexA sites on the ADE22 guide-donor plasmid and LexA-Fkh1p on a plasmid harboring Cas9-induced double strand breaks.

In order to validate MAGESTIC's high-throughput capacity, the team designed a guide-donor library to saturate a region of the essential eukaryotic gene Sec14 with amino acid substitutions. Believing that saturation mutagenesis could provide a complete map of residues important for Sec14p drug interactions, the team identified amino acid substitutions critical for chemical inhibition of lipid signaling, including Y111 variations and P120.

According to the study's authors, the substitutions potentially impair Sec14-inhibitor interactions by adjusting conformational changes that guide the inhibitors' entry into the Sec14p lipid binding pocket.

The team also used the MAGESTIC technique to insert thousands of natural single-nucleotide variants and indels without requiring protospacer adjacent motif (PAM) mutations. They found that single-nucleotide polymorphisms (SNPs) can be tolerated along the guide region at the genome scale, with a significant drop between the 19th and 20th positions from PAM. Analyzing the mismatch tolerance of 23,866 distinct guide-donor pairs across the genome, the team saw that a substantial fraction of donors with SNPs throughout the guide target region could be useful for editing and subsequent resistance to Cas9 cleavage.

The researchers also demonstrated that that Pol III terminating T-stretches play an important role in dictating guide efficacy in yeast by effectively reducing guide efficiency. Smith and his colleagues believe that using different promoters — such as Pol II promoters with ribozymes and poly(A) elements — may solve the issue of delivering guides from the Pol II promoter to T-rich genomic targets.

In an email, Alejandro Chavez, an assistant professor of pathology and cell biology at Columbia University who was not affiliated with the study but was part of a team that developed its own high-throughput gene editing methods in yeast, pointed out that MAGESTIC's main issue is linked to the efficiency of selecting the correct genomic edit.

"[MAGESTIC] seems to suffer from a decent number of non-edited cells in [the] population, but this is likely an issue that they will be able to overcome," Chavez explained.

Chavez is also unsure how well the team's method will translate to other yeast species. Highlighting Candida albicans, Chavez noted that the species has much lower levels of homologous recombinations than S. cerevisiae, and that MAGESTIC's genome editing ability would need to be assessed for this and similar species.

The Stanford team, however, believes that the MAGESTIC technique outperforms several limitations of currently available methods, including plasmid barcode instability and the inability to distinguish between oligo synthesis errors and sequencing errors in the guide and donor DNA during phenotyping.

"We can design variants that we want to make in the genome, and with proper guide donor design, we can generate a substantial fraction of designed variants genomewide" explained Kevin Roy, first author on the Nature Biotech paper and a postdoc in Stanford's genetics department. "By performing a study confirmation of each edit, we can conclude that we can create thousands of variants that are barcoded, and by only sequencing these short barcodes, we can track their effects all over the genome."

Compared to other CRISPR-editing techniques, Roy emphasized that that MAGESTIC can separate guide-donor sequence validation from variant quantification by tagging each guide-donor with the unique short barcode during cloning. In addition, he highlighted that MAGESTIC easily integrates the plasmid barcode into the yeast genome and removes residual guide-donor plasmid through plasmid self-destruction.

According to Lars Steinmetz, director of the Stanford Genome Technology Center and genetics professor at Stanford, and a co-author of the recently published study, MAGESTIC also differs from standard gene-editing methods because researchers do not have "to perform linkage or association before we go directly to the final step of validation." With MAGESTIC, researchers can instead perform "an allele replacement for every SNP in the genome and associate that with phenotypic changes that we directly measure."

While the researchers recently filed for a patent for the MAGESTIC technique, they do not have any current plans to commercialize the method. However, they are actively searching for licensees interested in utilizing the technology.

In the future, the researchers believe that MAGESTIC will be useful to discover the genetic basis of phenotypes in yeast species. Steinmetz envisions using the high-throughput system to examine the causal relationship between genes and phenotypic diversity.

In addition, Steinmetz aims to increase donor recruitment to boost rates of homologous recombination in mammalian cells, where survival rates of cells that experience double-strand breaks are much lower. He highlighted that a technology "like active donor recruitment, which pulls donor DNA to the site of the break, should help getting allele replacements or precision edits into a mammalian cell."

Robert St. Onge, co-author and senior scientist at the Stanford Genome Technology Center, noted that the team will use the technology for applications involving metabolic engineering.

"If something is producing a metabolite that you want, you may want to improve it somehow," St. Onge explained. "Knowing the exact genetic changes that are needed to make that improvement [and] having technology that gives you multiple shots on goal is really advantageous."

"Because we're designing the variants on the computer, we can make sure we're looking at all the substitutions that can be made and therefore go beyond what random mutagenesis can do," Steinmetz added. "Our tech allowed us to add replacements in the open reading frame that not only confer resistance to the drug, but also confer susceptibility to a drug in the screening approach."

Chavez believes that the MAGESTIC technique will be useful in the research space to better understand genome function —focusing on non-coding regions in the genome — along with characterizing proteins at "exquisite detail." In terms of the clinical space, he sees researchers potentially using the technique to model human genetic variation in simple systems like yeast, as well as performing comparisons at "library scales, [including] thousands of human variants created and characterized within a single set of experiments."