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RNA-guided gene drives for inheritance bias in yeast: Safe, responsible genome editing

Researchers encourage awareness and precaution as they use gBlocks® Gene Fragments to build gene drive constructs in S. cerevisiae

DiCarlo JE, Chavez A, et al. (2015) Safeguarding CRISPR-Cas9 gene drives in yeast. Nat Biotechnol, 33 (12):125–1255.

Background

Gene drives are genetic elements that persist in populations regardless of the effects they have on an organism’s reproductive fitness. Gene drives spread and are maintained through inheritance bias, a strategy commonly used by many organisms in nature. Most often, an inheritance-biasing gene functions by cleaving a homologous chromosome, which induces the cellular repair process to copy the gene and insert it into the damaged chromosome through homologous recombination. This mechanism, known as “homing”, has been well documented in S. cerevisiae, where the gene I-SceI encodes an endonuclease that cuts a gene encoding an rRNA subunit of the S. cerevisiae mitochondria.

While natural endonuclease homing systems like that in I-SceI are extremely efficient (I-SceI is correctly copied 99% of the time), it has been difficult for scientists to apply the mechanism to sequences with research applications. However, due to the recent advances in CRISPR-Cas9 technology, scientists can now target sequences with extremely high precision using guide RNA (gRNA) molecules. This ability raises questions as to whether Cas9 can, and should, be used to bias inheritance in eukaryotic organisms. In this paper, the authors investigate the feasibility of lab-driven eukaryotic inheritance bias. They accomplish this by building synthetic, Cas9-based gene drives in S. cerevisiae and measuring gene drive persistence through wild populations.

Importantly, this paper explains why including proper safeguards in gene drive experiments is necessary to control the potential impact of gene drive-containing organisms on natural ecological systems. To support this notion, the authors outline easy-to-use molecular confinement methods, and urge scientists to use them in future gene drive studies.

Methods

In their experiments, DiCarlo et al. took advantage of the red color that accumulates in yeast lacking functional copies of the ADE2 gene (yeast possessing at least 1 functional copy are cream-colored).

They first built a gene drive construct targeting ADE2 by replacing the endogenous ADE2 gene with a gRNA that targeted the wild-type ADE2 gene. They then mated these haploid, modified yeast to wild-type haploids in the presence and absence of the Cas9 plasmid and selected for diploids. They measured the persistence of the gene drive by observing the color of the resulting diploids, and used subsequent diploid dissection and sequencing to verify the colorimetric data.

The scientists also investigated whether RNA-guided gene drives could bias inheritance of a closely associated “cargo” gene in addition to the basic drive element. To do this, they repeated their earlier experiment, this time adding the URA3 gene in cis to the basic ADE2 drive element. They mated these URA3-containing drive haploids to wild-type haploids in the presence of Cas9 and selected for diploids again.

Finally, the group mated haploids containing the ADE2 gene drive to 6 phylogenetically and phenotypically diverse native S. cerevisiae strains. In these crosses, they quantitatively measured the efficiency of gene drive copying with qPCR.

In these experiments, the authors were required to build numerous gene drive constructs. All of the constructs in this research were synthesized using IDT gBlocks® Gene Fragments.

Results

Following the first cross (ADE2-gene drive containing haploids x wild type), nearly 100% of S. cerevisiae diploid colonies were red when Cas9 was present. This indicated highly efficient persistence of the ADE2 gene drive, which successfully cut the native ADE2 gene copy inherited from the wild-type parent. The scientists proceeded to sporulate the diploids and examine their haploid progeny. They found a perfect 4:0 ratio of red:cream haploids in all 18 dissected diploids, confirming that all copies of the native ADE2 locus were disrupted. They also sequenced the 72 haploids (derived from the 18 diploids) and discovered that all haploids contained intact ADE2 gene drives with no additional mutations.

The group observed similar results when they examined the persistence of the "cargo" gene, URA3. URA3 allows yeast to grow in the absence of uracil. They crossed in cis haploids containing URA3 with wild type and found that all of the progeny grew normally on uracil-deficient media, indicating that both the gene and gene drive were efficiently copied. Again, the scientists dissected 18 diploids and found that all sporulated haploid cells were red, confirming successful persistence of the ADE2 gene drive.

As with the previous experiments, the authors saw extremely high efficiency when haploids containing the ADE2 gene drive were crossed with phylogenetically diverse S. cerevisiae strains. qPCR confirmed that over 99% of the diploid chromosomes contained the gene drive regardless of wild-type parent.

This research represents the first example of an inheritance-biasing synthetic endonuclease gene drive. While the robustness of these findings are encouraging for potential applications, the authors note that gene drives must be handled safely and responsibly due to their potential impact on natural ecological systems. To eliminate any possibility of RNA-guided gene drives spreading in the wild, they advise scientists to employ the molecular confinement methods outlined in this paper. They also urge collaborative, precautionary discussions with the general public in advance of any real-world gene drive applications.

References

Authors at the Wyss Institute. FAQs: Gene drives. The Wyss Institute for Biologically Inspired Engineering at Harvard University. (Accessed February 24–25, 2016.)

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Custom dsDNA fragments

gBlocks® Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–2000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.


Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.

Related reading

Getting started with Alt-R® CRISPR-Cas9 genome editing—Learn about the components of the Alt‑R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

5 pieces of data that will change how you set up your CRISPR-Cas9 experiments—To improve the efficiency of CRISPR-Cas9 genome editing, IDT scientists evaluated several factors that influence how we design and perform genome editing experiments. Review the data and results for 5 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

The gene construction revolution—See how use of high-quality, custom dsDNA fragments as a starting material allows you to turn what might otherwise be multi-step cloning assemblies into simpler reactions. You can often just order an entire target sequence that is ready for cloning or other uses.

iGEM students engineer biological tools for a better world—Projects from 2 of the prize-winning 2013 iGEM teams show how non-standard natural and synthetic amino acids can be used in 1) peptide synthesis, and 2) tuberculosis monitoring and treatment. Both projects make use of gBlocks Gene Fragments to speed up construct assembly.

Codon optimization tool makes synthetic genes easy—Use the IDT Codon Optimization Tool to simplify designing synthetic genes and gBlocks® Gene Fragments for expression in a variety of organisms.

Author: Nolan Speicher is a scientific writer at IDT.

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