Bio


I am a postdoc in the lab of Lars Steinmetz. My research primarily focuses on genome engineering and CRISPR technology development. Specifically I work on technology development for high throughput precision genome editing including working on technologies to improve the efficiency of homologous recombination repair. I also worked on developing a robust CRISPRi system for yeast, and studying the effects of guide-target mismatches on Cas9 cleavage efficacy.

Honors & Awards


  • Outstanding Achievement Award in Molecular Biology, UC San Diego (June 2006)
  • Stanford Graduate Fellowship, Stanford University (Sept 2010)

Professional Education


  • Bachelor of Science, University of California San Diego, Molecular Biology (2006)
  • Doctor of Philosophy, Stanford University, GENE-PHD (2016)

Lab Affiliations


All Publications


  • Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9 NATURE MICROBIOLOGY Fu, B., Smith, J. D., Fuchs, R. T., Mabuchis, M., Curcuru, J., Robb, G., Fire, A. Z. 2019; 4 (5): 888–97
  • Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nature biotechnology Roy, K. R., Smith, J. D., Vonesch, S. C., Lin, G., Tu, C. S., Lederer, A. R., Chu, A., Suresh, S., Nguyen, M., Horecka, J., Tripathi, A., Burnett, W. T., Morgan, M. A., Schulz, J., Orsley, K. M., Wei, W., Aiyar, R. S., Davis, R. W., Bankaitis, V. A., Haber, J. E., Salit, M. L., St Onge, R. P., Steinmetz, L. M. 2018

    Abstract

    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 we 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. We 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. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We 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.

    View details for PubMedID 29734294

  • Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo NUCLEIC ACIDS RESEARCH Fu, B. X., Onge, R. P., Fire, A. Z., Smith, J. D. 2016; 44 (11): 5365-5377

    View details for DOI 10.1093/nar/gkw417

    View details for Web of Science ID 000379753100041

  • Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome biology Smith, J. D., Suresh, S., Schlecht, U., Wu, M., Wagih, O., Peltz, G., Davis, R. W., Steinmetz, L. M., Parts, L., St Onge, R. P. 2016; 17 (1): 45-?

    Abstract

    Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

    View details for DOI 10.1186/s13059-016-0900-9

    View details for PubMedID 26956608

    View details for PubMedCentralID PMC4784398

  • Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation. PLoS genetics Naranjo, S., Smith, J. D., Artieri, C. G., Zhang, M., Zhou, Y., Palmer, M. E., Fraser, H. B. 2015; 11 (12)

    Abstract

    Although single genes underlying several evolutionary adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. Here we report that the budding yeast Saccharomyces paradoxus has recently evolved resistance to citrinin, a naturally occurring mycotoxin. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in the citrinin response that are constitutively up-regulated in S. paradoxus. Four of these genes are necessary for resistance, and are also sufficient to increase the resistance of a sensitive strain when over-expressed. Moreover, cis-regulatory divergence in the promoters of these genes contributes to resistance, while exacting a cost in the absence of citrinin. Our results demonstrate how the subtle effects of individual regulatory elements can be combined, via natural selection, into a complex adaptation. Our approach can be applied to dissect the genetic basis of polygenic adaptations in a wide range of species.

    View details for DOI 10.1371/journal.pgen.1005751

    View details for PubMedID 26713447

  • A Novel Test for Selection on cis-Regulatory Elements Reveals Positive and Negative Selection Acting on Mammalian Transcriptional Enhancers. Molecular biology and evolution Smith, J. D., McManus, K. F., Fraser, H. B. 2013; 30 (11): 2509-2518

    Abstract

    Measuring natural selection on genomic elements involved in the cis-regulation of gene expression-such as transcriptional enhancers and promoters-is critical for understanding the evolution of genomes, yet it remains a major challenge. Many studies have attempted to detect positive or negative selection in these noncoding elements by searching for those with the fastest or slowest rates of evolution, but this can be problematic. Here, we introduce a new approach to this issue, and demonstrate its utility on three mammalian transcriptional enhancers. Using results from saturation mutagenesis studies of these enhancers, we classified all possible point mutations as upregulating, downregulating, or silent, and determined which of these mutations have occurred on each branch of a phylogeny. Applying a framework analogous to Ka/Ks in protein-coding genes, we measured the strength of selection on upregulating and downregulating mutations, in specific branches as well as entire phylogenies. We discovered distinct modes of selection acting on different enhancers: although all three have experienced negative selection against downregulating mutations, the selection pressures on upregulating mutations vary. In one case, we detected positive selection for upregulation, whereas the other two had no detectable selection on upregulating mutations. Our methodology is applicable to the growing number of saturation mutagenesis data sets, and provides a detailed picture of the mode and strength of natural selection acting on cis-regulatory elements.

    View details for DOI 10.1093/molbev/mst134

    View details for PubMedID 23904330

    View details for PubMedCentralID PMC3808868

  • Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Tang, M., Fischer, C. R., Chari, J. V., Tan, D., Suresh, S., Chu, A., Miranda, M., Smith, J., Zhang, Z., Garg, N. K., St Onge, R. P., Tang, Y. 2019; 141 (20): 8198–8206

    Abstract

    Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATP-independent fashion. The KU42 TE domain selects cysteine and homocysteine and catalyzes transthioesterification using the side-chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcases the functional diversity of HRPKS enzymes and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.

    View details for DOI 10.1021/jacs.9b01083

    View details for Web of Science ID 000469292300028

    View details for PubMedID 31051070

  • Improved discovery of genetic interactions using CRISPRiSeq across multiple environments GENOME RESEARCH Jaffe, M., Dziulko, A., Smith, J. D., St Onge, R. P., Levy, S. F., Sherlock, G. 2019; 29 (4): 668–81
  • Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9. Nature microbiology Fu, B. X., Smith, J. D., Fuchs, R. T., Mabuchi, M., Curcuru, J., Robb, G. B., Fire, A. Z. 2019

    Abstract

    Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.

    View details for PubMedID 30833733

  • Improved discovery of genetic interactions using CRISPRiSeq across multiple environments. Genome research Jaffe, M., Dziulko, A., Smith, J. D., St Onge, R. P., Levy, S. F., Sherlock, G. 2019

    Abstract

    Large-scale Genetic Interaction (GI) screens in yeast have been invaluable for our understanding of molecular systems biology, and for characterizing novel gene function. Due in part to the high costs and long experiment times required, a preponderance of GI data has been generated in a single environmental condition. However, an unknown fraction of GIs may be specific to other conditions. Here, we developed a pooled-growth CRISPRi-based sequencing assay for genetic interactions, CRISPRiSeq, that increases throughput such that GIs can be easily assayed across multiple growth conditions. We assayed the fitness of ~17,000 strains encompassing ~7,700 pairwise interactions in five conditions and found that the additional conditions increased the number of GIs detected nearly 3-fold over the number detected in rich media alone In addition, we found that condition-specific GIs are prevalent and improved the power to functionally classify genes. Finally, we found new links during respiratory growth between members of the Ras-nutrient sensing pathway and both the COG complex and a gene of unknown function. Our results highlight the potential of conditional GI screens to improve our understanding of cellular genetic networks.

    View details for PubMedID 30782640

  • Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing. Cell Sharon, E., Chen, S. A., Khosla, N. M., Smith, J. D., Pritchard, J. K., Fraser, H. B. 2018

    Abstract

    A major challenge in genetics is to identify genetic variants driving natural phenotypic variation. However, current methods of genetic mapping havelimited resolution. To address this challenge, we developed aCRISPR-Cas9-based high-throughput genome editing approach that can introduce thousands of specific genetic variants in a single experiment. This enabled us to study the fitness consequences of 16,006 natural genetic variants in yeast. We identified 572 variants with significant fitness differences in glucose media; these are highly enriched in promoters, particularly in transcription factor binding sites, while only 19.2% affect amino acid sequences. Strikingly, nearby variants nearly always favor the same parent's alleles, suggesting that lineage-specific selection is often driven by multiple clusteredvariants. In sum, our genome editing approach reveals the genetic architecture of fitness variation at single-base resolution and could be adapted tomeasure the effects of genome-wide genetic variation in any screen for cell survival or cell-sortable markers.

    View details for PubMedID 30245013

  • Quantitative analysis of protein interaction network dynamics in yeast MOLECULAR SYSTEMS BIOLOGY Celaj, A., Schlecht, U., Smith, J. D., Xu, W., Suresh, S., Miranda, M., Aparicio, A., Proctor, M., Davis, R. W., Roth, F. P., St. Onge, R. P. 2017; 13 (7): 934

    Abstract

    Many cellular functions are mediated by protein-protein interaction networks, which are environment dependent. However, systematic measurement of interactions in diverse environments is required to better understand the relative importance of different mechanisms underlying network dynamics. To investigate environment-dependent protein complex dynamics, we used a DNA-barcode-based multiplexed protein interaction assay in Saccharomyces cerevisiae to measure in vivo abundance of 1,379 binary protein complexes under 14 environments. Many binary complexes (55%) were environment dependent, especially those involving transmembrane transporters. We observed many concerted changes around highly connected proteins, and overall network dynamics suggested that "concerted" protein-centered changes are prevalent. Under a diauxic shift in carbon source from glucose to ethanol, a mass-action-based model using relative mRNA levels explained an estimated 47% of the observed variance in binary complex abundance and predicted the direction of concerted binary complex changes with 88% accuracy. Thus, we provide a resource of yeast protein interaction measurements across diverse environments and illustrate the value of this resource in revealing mechanisms of network dynamics.

    View details for PubMedID 28705884

  • Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies MICROBIAL CELL FACTORIES Jensen, E. D., Ferreira, R., Jakociunas, T., Arsovska, D., Zhang, J., Ding, L., Smith, J. D., David, F., Nielsen, J., Jensen, M. K., Keasling, J. D. 2017; 16

    Abstract

    Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.

    View details for DOI 10.1186/s12934-017-0664-2

    View details for PubMedID 28298224

  • A method for high-throughput production of sequence-verified DNA libraries and strain collections. Molecular systems biology Smith, J. D., Schlecht, U., Xu, W., Suresh, S., Horecka, J., Proctor, M. J., Aiyar, R. S., Bennett, R. A., Chu, A., Li, Y. F., Roy, K., Davis, R. W., Steinmetz, L. M., Hyman, R. W., Levy, S. F., St Onge, R. P. 2017; 13 (2): 913-?

    Abstract

    The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

    View details for DOI 10.15252/msb.20167233

    View details for PubMedID 28193641

    View details for PubMedCentralID PMC5327727

  • Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo. Nucleic acids research Fu, B. X., St Onge, R. P., Fire, A. Z., Smith, J. D. 2016; 44 (11): 5365-5377

    Abstract

    Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.

    View details for DOI 10.1093/nar/gkw417

    View details for PubMedID 27198218

    View details for PubMedCentralID PMC4914125

  • Dissecting the Genetic Basis of a Complex cis-Regulatory Adaptation PLOS GENETICS Naranjo, S., Smith, J. D., Artieri, C. G., Zhang, M., Zhou, Y., Palmer, M. E., Fraser, H. B. 2015; 11 (12)

    Abstract

    Although single genes underlying several evolutionary adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. Here we report that the budding yeast Saccharomyces paradoxus has recently evolved resistance to citrinin, a naturally occurring mycotoxin. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in the citrinin response that are constitutively up-regulated in S. paradoxus. Four of these genes are necessary for resistance, and are also sufficient to increase the resistance of a sensitive strain when over-expressed. Moreover, cis-regulatory divergence in the promoters of these genes contributes to resistance, while exacting a cost in the absence of citrinin. Our results demonstrate how the subtle effects of individual regulatory elements can be combined, via natural selection, into a complex adaptation. Our approach can be applied to dissect the genetic basis of polygenic adaptations in a wide range of species.

    View details for DOI 10.1371/journal.pgen.1005751

    View details for Web of Science ID 000368518400074

  • Whole-genome sequencing of the world's oldest people. PloS one Gierman, H. J., Fortney, K., Roach, J. C., Coles, N. S., Li, H., Glusman, G., Markov, G. J., Smith, J. D., Hood, L., Coles, L. S., Kim, S. K. 2014; 9 (11)

    Abstract

    Supercentenarians (110 years or older) are the world's oldest people. Seventy four are alive worldwide, with twenty two in the United States. We performed whole-genome sequencing on 17 supercentenarians to explore the genetic basis underlying extreme human longevity. We found no significant evidence of enrichment for a single rare protein-altering variant or for a gene harboring different rare protein altering variants in supercentenarian compared to control genomes. We followed up on the gene most enriched for rare protein-altering variants in our cohort of supercentenarians, TSHZ3, by sequencing it in a second cohort of 99 long-lived individuals but did not find a significant enrichment. The genome of one supercentenarian had a pathogenic mutation in DSC2, known to predispose to arrhythmogenic right ventricular cardiomyopathy, which is recommended to be reported to this individual as an incidental finding according to a recent position statement by the American College of Medical Genetics and Genomics. Even with this pathogenic mutation, the proband lived to over 110 years. The entire list of rare protein-altering variants and DNA sequence of all 17 supercentenarian genomes is available as a resource to assist the discovery of the genetic basis of extreme longevity in future studies.

    View details for DOI 10.1371/journal.pone.0112430

    View details for PubMedID 25390934

    View details for PubMedCentralID PMC4229186

  • Whole-genome sequencing of the world's oldest people. PloS one Gierman, H. J., Fortney, K., Roach, J. C., Coles, N. S., Li, H., Glusman, G., Markov, G. J., Smith, J. D., Hood, L., Coles, L. S., Kim, S. K. 2014; 9 (11)

    View details for DOI 10.1371/journal.pone.0112430

    View details for PubMedID 25390934

  • Evolution of programmable zinc finger-recombinases with activity in human cells JOURNAL OF MOLECULAR BIOLOGY Gordley, R. M., Smith, J. D., Graslund, T., Barbas, C. F. 2007; 367 (3): 802-813

    Abstract

    Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (Rec(ZF)s) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in approximately 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)s may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.

    View details for DOI 10.1016/j.jmb.2007.01.017

    View details for Web of Science ID 000245123700017

    View details for PubMedID 17289078