Multiplexed precision genome editing with trackable genomic barcodes in yeast

Content introduction:

  • High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
  • Multiplexed precision genome editing with trackable genomic barcodes in yeast
  • Secure genome-wide association analysis using multiparty computation
  • Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision
  • Reversal of siRNA-mediated gene silencing in vivo

1. High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, Xiaoge Guo at Wyss Institute for Biologically Inspired Engineering at Harvard University in Boston, Massachusetts, USA and his colleagues introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. They demonstrate the utility of their approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, they created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Their strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

Read more, please click https://www.nature.com/articles/nbt.4147

2. Multiplexed precision genome editing with trackable genomic barcodes in yeast
Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here Kevin R Roy at Stanford University in Palo Alto, California, USA and his colleagues describe a CRISPR–Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide–donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. They demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA–Fkh1p fusion protein. They performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. They also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

Read more, please click https://www.nature.com/articles/nbt.4137

3. Secure genome-wide association analysis using multiparty computation
Most sequenced genomes are currently stored in strict access-controlled repositories. Free access to these data could improve the power of genome-wide association studies (GWAS) to identify disease-causing genetic variants and aid the discovery of new drug targets. However, concerns over genetic data privacy may deter individuals from contributing their genomes to scientific studies and could prevent researchers from sharing data with the scientific community. Although cryptographic techniques for secure data analysis exist, none scales to computationally intensive analyses, such as GWAS. Here Hyunghoon Cho at Massachusetts Institute of Technology in Cambridge, Massachusetts, USA and his colleagues describe a protocol for large-scale genome-wide analysis that facilitates quality control and population stratification correction in 9K, 13K, and 23K individuals while maintaining the confidentiality of underlying genotypes and phenotypes. They show the protocol could feasibly scale to a million individuals. This approach may help to make currently restricted data available to the scientific community and could potentially enable secure genome crowdsourcing, allowing individuals to contribute their genomes to a study without compromising their privacy.

Read more, please click https://www.nature.com/articles/nbt.4108

4. Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision
Zehua Bao at University of Illinois at Urbana-Champaign in Urbana, Illinois, USA and his colleagues developed a CRISPR–Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. They validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply their method to improve tolerance to growth inhibitors.

Read more, please click https://www.nature.com/articles/nbt.4132

5. Reversal of siRNA-mediated gene silencing in vivo
Ivan Zlatev at Alnylam Pharmaceuticals in Cambridge, Massachusetts, USA and his colleagues report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. They found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Their modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc–siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.

Read more, please click https://www.nature.com/articles/nbt.4136

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