scholarly journals CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

2019 ◽  
Author(s):  
Zhiliang Yang ◽  
Harley Edwards ◽  
Peng Xu
Keyword(s):  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Standard Type ◽  
Editing Efficiency ◽  
Color Changes ◽  
Indel Mutation ◽  
Protospacer Adjacent Motif ◽  
Colony Color ◽  
First Time

AbstractCRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5’-phosphate decarboxylase (∼96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3’ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.

scholarly journals Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

2019 ◽  
Author(s):  
Zhuqing Wang ◽  
Yue Wang ◽  
Shawn Wang ◽  
Andrew J Gorzalski ◽  
Hayden McSwiggin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Editing Efficiency ◽  
Protospacer Adjacent Motif ◽  
Summary Statement ◽  
Strict Requirement ◽  
Moraxella Bovoculi ◽  
Target Effects

AbstractDespite many advantages over Cas9, Cas12a has not been widely used in genome editing in mammalian cells largely due to its strict requirement of the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM sequences and with minimal on-target mutations and close to 100% editing efficiency when crRNAs of 23nt spacers were used.Summary statementCRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and high overall knock-in efficiency.

scholarly journals Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

2020 ◽  
Author(s):  
Wei Wang ◽  
Bin Tian ◽  
Qianli Pan ◽  
Yueying Chen ◽  
Fei He ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Genome Editing ◽  
Agronomic Traits ◽  
Wheat Genome ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Endogenous Genes

AbstractThe development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

scholarly journals Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianhang Wang ◽  
Mingxing Tu ◽  
Ya Wang ◽  
Wuchen Yin ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Editing ◽  
Genome Sequencing ◽  
Plant Biotechnology ◽  
High Specificity ◽  
Fruit Trees ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Indel Mutation ◽  
Target Sites

AbstractThe CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

scholarly journals Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Pratik Prashant Pawar ◽  
Annamma Anil Odaneth ◽  
Rajeshkumar Natwarlal Vadgama ◽  
Arvind Mallinath Lali
Keyword(s):  
Yarrowia Lipolytica ◽  
Oil Recovery ◽  
Oleaginous Yeast ◽  
Fermentation Broth ◽  
Continuous Production ◽  
Oil Production ◽  
Biofuel Production ◽  
Oil Yield ◽  
Microbial Oil

Abstract Background Recent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (Heff/C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process. Results Adsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration. Conclusion The nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.

scholarly journals AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liyang Zhang ◽  
John A. Zuris ◽  
Ramya Viswanathan ◽  
Jasmine N. Edelstein ◽  
Rolf Turk ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Genome Editing ◽  
Nk Cell ◽  
Basic Research ◽  
Cell Recognition ◽  
Site Specific ◽  
Editing Efficiency ◽  
Rapid Generation ◽  
Therapeutic Cell

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

scholarly journals Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sivamoke Dissook ◽  
Tomohisa Kuzuyama ◽  
Yuri Nishimoto ◽  
Shigeru Kitani ◽  
Sastia Putri ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Isoprenoid Biosynthesis ◽  
Mep Pathway ◽  
Authentic Standard ◽  
Chemical Inhibition ◽  
Chromatographic Peaks ◽  
Methyl Erythritol Phosphate ◽  
Limiting Condition ◽  
Yeast Yarrowia Lipolytica

AbstractMethyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

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