Inefficient homology-directed repair (HDR) constrains CRISPR–Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform—lowered indel nuclease system enabling accurate repair (LINEAR)—which enhanced HDR rates (to 67–100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.
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Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris
Microbial Cell Factories Open Access 07 September 2022
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Source data are provided for the results presented in Main and Extended Data figures. Vectors containing LINEAR constructs for Y. lipolytica, K. marxianus, H. polymorpha and S. stipitis are available from Addgene (plasmid nos. 174837–174840). Raw reads and assembled genome sequences for the final engineered norcoclaurine-producing strains have been deposited in NCBI under BioProject ID PRJNA753835. Additional data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
All code used for processing and assembly of genome sequencing reads (along with an accompanying method for how the code was implemented) is available on Zenodo: https://doi.org/10.5281/zenodo.5544230.
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This work was supported by the National Science Foundation Grant (no. 1716837 to D.P., Y.Z., M.C., S.G., C.L., M.S., S.C., A.S., L.H. and Z.S.), the NSF Graduate Research Fellowship Program (to D.P.) and Griswold Internship (to M.G.). We thank T. W. Jeffries (professor emeritus, University of Wisconsin-Madison and the Founder of Xylome Corporation) for sharing S. stipitis FLP-UC7 (ura3-3, NRRL Y21448) and the plasmid pJML545. We thank I. Wheeldon, University of California at Riverside, for sharing K. marxianus YS402 (CBC6556 Δura3); J. Dueber, University of California at Berkeley for sharing the S. cerevisiae plasmid containing TyrH* and DOD; and S. Yang, Chinese Academy of Science, for sharing the Y. lipolytica plasmids pCas1-yl and pCAS2-yl. We also thank S. M. Rigby for flow cytometry and L. Showman and K. Narayanaswamy at the W. M. Keck Metabolomics Research Laboratory of ISU for the detection of (S)-norcoclaurine. Finally, we thank T. Murtha at the ISU DNA facility for preparing gDNA samples for Nanopore sequencing.
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Zihe Liu, Eric Young and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Literature survey to determine the most implemented CRISPR platform.
A compilation of 400 instances where studies deployed CRISPR for genome editing revealed that most researchers implement the conventional CRISPR plasmid and donor DNA Platform I. Studies were identified on PubMed using ‘Yeast CRISPR’ and ‘Mammal CRISPR’ in the search query.
Extended Data Fig. 2 The shortcomings associated with the conventional CRISPR platforms in S. stipitis.
a, The workflow for the conventional CRISPR plasmid and donor DNA platform (Platform I in Extended Data Fig. 1) deployed in S. stipitis, depicting various shortcomings our group has encountered that impeded obtaining correctly edited cells. b, The vector map of the consolidated ‘self-digesting’ plasmid, pCashGem-SD-Xyl2, highlighting the key design components (Platform IV in Extended Data Fig. 1). c, Transformation of UC7 with the pCashGem-SD-Xyl2 plasmid failed to address the shortcomings, with almost all the colonies displaying an episomal GFP expression profile.
Extended Data Fig. 3 Aberrant recombination characterization in S. stipitis.
a, Visual depiction of visibly intense GFP expression and subsequent loss of expression b, Vector map of the pGAOU plasmid, harboring the GAOU fragment flanked by SacI and XhoI restriction sites. The digested GAOU fragment was transformed into UC7. c, Ten strongly intense green colonies observed under the DR46B transilluminator. d, The GFP expression intensity of the ten variants was evaluated by flow cytometry after 36 h of cultivation. Numbers correspond to the mean GFP expression intensity. Among these ten variants, only #5, #7, and #9 exhibited genomic expression of GFP (sharp peaks). The gating methodology was summarized in Supplementary Information.
Extended Data Fig. 4 Verification of ‘pseudo’ plasmids formed by aberrant recombination events in S. stipitis.
a, Transformation of E. coli BW25141 with the five plasmids isolated from S. stipitis variants. b, Enzyme digestion verification of pGAOU-X vectors isolated from E. coli DH5α. Lane 1: M, GeneRuler 1 kb DNA Ladder. Lanes 2 and 3: pGAOU-1 isolates. Lanes 4 and 5: pGAOU-2 isolates. Lanes 6 and 7: pGAOU-3 isolates. Individual restriction digestions for each biological sample were performed once. c, Vector maps of the isolated pGAOU-X plasmids. d, Transformation of UC7 with pGAOU-2 and pGAOU-3. Plates were observed under a DR46B transilluminator.
Extended Data Fig. 5 Design protocol for the LINEAR platform.
a, Cloning of individual LINEAR components. b, Assembly of LINEAR components into a cloning vector. Digestion and gel electrophoresis of the assembled vector yields the LINEAR fragment used in genome editing.
Extended Data Fig. 6 Comparison of the conventional CRISPR platforms versus LINEAR highlights growth deficiency associated with constitutive Cas9 expression.
All platforms utilized hGem tagged-Cas9. Plate pictures were taken at various timepoints post-transformation.
Extended Data Fig. 7 Assessing the contribution of the hGem tag on Xyl2:URA3 LINEAR editing efficiency with 500-bp homology arms.
a, Equimolar quantities of each LINEAR fragment were transformed to S. stipitis UC7. Plate pictures were taken at 48 h post-transformation. Ten colonies were selected at random (circled) for downstream analysis. b, The expected edit outcome on Xyl2 locus. c, PCR products obtained from the primer pair in b using the genomic DNA isolated from the colonies circled in a as templates. Individual PCR reactions for each biological sample were performed once. d, Sequencing of the amplicon associated with Cas9 #4 revealed an erroneous insertion of the donor template at the 5’ end of Xyl2.
Extended Data Fig. 8 Assessing the contribution of the hGem tag on Xyl2:URA3 LINEAR editing efficiency with 50-bp homology arms.
a, Equimolar quantities of each LINEAR fragment were transformed to S. stipitis UC7. Plate pictures were taken at 48 h post-transformation. Ten colonies selected at random (circled) for downstream analysis. Note that GFP cassette was placed in between sgRNA cassette and the donor DNA as a negative reporter to assess aberrant recombination. The colonies with strong GFP expression levels were false positives. b, The expected edit outcome on Xyl2 locus and the intact locus. c, PCR products obtained from the primer pair in b using the genomic DNA isolated from the colonies circled in a as templates. Individual PCR reactions for each biological sample were performed once. d, Sequencing of the amplicons associated with Cas9-hGem #10 and Cas9 #6 confirmed the presence of the intact wild type Xyl2. e, Sequencing of the amplicon associated with Cas9 #2 revealed an erroneous insertion of the donor template along with a portion of the upstream region of the LINEAR fragment.
Extended Data Fig. 9 Assessing the contribution of the hGem tag on Xyl2:GSAU LINEAR editing efficiency.
a, Equimolar quantities of each LINEAR fragment were transformed to S. stipitis UC7. Plate pictures were taken at 48 h post-transformation. Ten colonies that displayed genomic GFP expression as visualized on transilluminator selected (circled) for downstream analysis. Slightly different from the design used to achieve Xyl2:URA3 LINEAR editing with 50-bp homology arms, GFP cassette was included in the donor DNA as a positive reporter. b, PCR analysis of the 5’ end of the Xyl2 locus. Due to the large size of the inserted pathway, two reactions were designed to assess the nature of integration at each 500-bp homology arm. PCR products were obtained from the primer pair using the genomic DNA isolated from the colonies circled in a as templates. Individual PCR reactions for each biological sample were performed once. c, PCR analysis of the 3’ end of the Xyl2 locus. Individual PCR reactions for each biological sample were performed once. d, Sequencing of the amplicon associated with Cas9 #9 revealed an erroneous insertion of the downstream portion of the donor template at the 3’ end of Xyl2. Wild type stands for a ~100-bp sequence originating from the wild type Xyl2 locus between the upstream and the downstream homology arms.
Extended Data Fig. 10 Pathway map depicting the aromatic amino acid pathway and the de novo synthesis of (S)-norcoclaurine.
a, Glycolysis and the pentose phosphate pathway (PPP) channel carbon fluxes into the aromatic amino acid (AAA) pathway. Shikimate can serve as a reporter for assessing AAA pathway flux. Tkt1, Aro4*, Aro1, and Aro7* comprise the four-enzyme, upstream L-tyrosine precursor module. The red lines depict L-tyrosine feedback inhibition on the native Aro4 and Aro7 enzymes. b, (S)-betalamic acid, a product of the biosensing module of TyrH* and DOD, spontaneously condenses with endogenous amino acids to produce yellow pigmented betaxanthins. c, TyrH*, DODC, and NCS comprise the three-enzyme, downstream (S)-norcoclaurine module. Metabolite abbreviations: G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; DHQ, 3-dehydroquinic acid; DHS, 3-dehydroshikimate; S3P, shikmate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; L-DOPA, L-3,4-dihydroxyphenylalanine; 4-HPAA, 4-hydroxyphenylacetaldehyde. Enzyme abbreviations: Tkt1, transketolase; Aro4*, DAHP synthase feedback inhibition-resistant mutant (Aro4K220L); Aro1, pentafunctional Aro1 polypeptide; Aro1*, Aro1 mutant (Aro1D900A) with the shikimate kinase domain inactivated, enabling shikimate accumulation; Aro7*, chorismate mutase feedback inhibition-resistant mutant (Aro7G139S); TyrH*, tyrosine hydroxylase mutant (TyrHW13L, F309L) from Berberis vulgaris with low L-DOPA oxidase activity; DOD, L-DOPA dioxygenase from Mirabilis jalapa; DODC, L-DOPA decarboxylase from Pseudomonas putida; NCS, norcoclaurine synthase from Papaver somniferum.
Supplementary Discussions 1–4, Figs. 1–22 and Tables 1–5.
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Ploessl, D., Zhao, Y., Cao, M. et al. A repackaged CRISPR platform increases homology-directed repair for yeast engineering. Nat Chem Biol 18, 38–46 (2022). https://doi.org/10.1038/s41589-021-00893-5
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Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris
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