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

Abstract

CRISPR-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.

An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing

“…This might be the result of the high accessibility of the ylAC linear DNA fragments, unlike those of most gene integration sites in native chromosomes. Higher HR rates of gene integration have been achieved using site-directed nucleases, such as TALEN 16 and CRISPR/Cas9 or Cas12a/Cpf1 15,18,51,52 , to introduce double-strand breaks (DSB) thus making the target integration site on the genome more accessible. Nevertheless, gene integration in natural chromosomes is highly dependent on the integration locus and on the size of homologous regions 28,29,39 , two limits that are eliminated when using the ylAC.…”

“…For decades, these desirable physiological traits and its "generally recognized as safe" (GRAS) status 5 , have made Y. lipolytica a strain of interest for the detergents, food, pharmaceutical and environmental industries 4,6,7 . Inevitably, industrial interest in Y. lipolytica has provided the impetus to develop a variety of basic genetic tools, including those for protein expression [8][9][10][11][12] , YaliBrick-based cloning 13 , iterative gene integration 14 and nuclease-based genome-editing [15][16][17][18] , etc. Combined with the release of the full genome sequence of Y. lipolytica (CLIB 122) in 2004 19 , these tools fuelled basic and applied research that has yielded modified strains capable of producing biofuels, pharmaceuticals, basic commodities and building blocks for the chemical industry [20][21][22] .…”

“…The recent introduction of nuclease-based strategies (e.g. Cas9, Cas12a/Cpf1 and TALEN) [16][17][18]39 has provided the means to overcome some of the above-mentioned limitations. However, HR still relies on the use of large homology regions of 1 kb or more to achieve satisfactory recombination efficiencies 40 .…”

Activation of pancreatic β-cell genes by multiplex epigenetic CRISPR-editing

“…On the contrary, the use of CRISPR/dCas9 associated with p300 can be used efficiently with a single sgRNA. 20 While CRISPR gene editing was extensively used as a multiplex platform for modifying several genes at the same time, [22][23][24][25][26][27][28][29][30][31][32] only few reports showed the multiplexing capacity of CRISPRa platform, 17,18,33 and none of them in a significant number of genes corresponding to the same cellular lineage.…”

Optimizing mevalonate pathway for squalene production inYarrowia lipolytica

“…It has also been recognized as a 'generally regarded as safe' (GRAS) organism (Groenewald et al, 2014) in the food and nutraceutical industry. A large collection of customized genetic toolboxes, including YaliBricks gene assembly (Wong, Engel, Jin, Holdridge, & Xu, 2017), CRISPR-Cas9 (Bae, Park, Kim, & Hahn, 2020;Macarena Larroude, Trabelsi, Nicaud, & Rossignol, 2020) or CRISPR-Cpf1 (Yang, Edwards, & Xu, 2020) genome editing, Cre-LoxP-based iterative chromosomal integrations (Lv, Edwards, Zhou, & Xu, 2019), transposon-based mutagenesis (Wagner, Williams, & Alper, 2018) and Golden-gate cloning (Celińska et al, 2017;Egermeier, Sauer, & Marx, 2019;M. Larroude et al, 2019), enabled us to rapidly modify its genome and evaluate many metabolic events to explore the catalytic diversity of this yeast beyond its regular portfolio of fatty acids, fatty alcohols, biofuels et al Recent metabolic engineering effort in this yeast has allowed us to access more specialized secondary metabolites with pharmaceutical values, including sesquiterpenes (Marsafari & Xu, 2020), triterpenoids (Jin et al, 2019) and flavonoids (Lv, Marsafari, et al, 2019;Palmer, Miller, Nguyen, & Alper, 2020) et al Isoprenoids are a large group of natural products with diverse biological functions.…”