Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

Baisya, Dipankar Ranjan; Ramesh, Adithya; Schwartz, Cory; Lonardi, Stefano; Wheeldon, Ian

doi:10.1038/s41467-022-28540-0

Cited by 33 publications

(46 citation statements)

References 51 publications

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“…The pooled guide libraries contain single guide RNAs (sgRNAs) that target more than 98.5% of the protein-coding sequences with 6-and 8-fold coverage for Cas9 and Cas12a, respectively. Guide activity in these libraries was previously reported 9,16 ; a cutting score (CS), defined as the - log 2 ratio of normalized read counts obtained in PO1f Cas9/12a ΔKU70 to counts in the control strain, was determined for each guide ( Fig. 1a ).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, we sought to test the performance of acCRISPR using computationally predicted sgRNA activity scores. Among the large set of guide prediction tools available for Cas9, we selected DeepGuide 16 , uCRISPR 24 , Designer v1 25 , Designer v2 26 , SSC 27 , CRISPRscan 28 , and CRISPRspec 29 ( Fig. 4 and Supplementary File 9 ).…”

Section: Resultsmentioning

confidence: 99%

“…sgRNA library design sgRNA library design for the Cas9 and Cas12a CRISPR systems was accomplished as previously described in refs. 9 and 16 . The critical elements of the design are described again here.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…The Cas12a library targeting the protein-coding genes in PO1f was ordered as an oligonucleotide pool from Agilent Technologies Inc. and cloned in-house using the Agilent SureVector CRISPR Library Cloning Kit (Part Number G7556A) as previously described in 16 .…”

Section: Sgrna Library Cloningmentioning

confidence: 99%

“…Using previously derived guide activity profiles of Yarrowia genome-wide Cas9 and -12a libraries (see ref. 16 ), along with new growth screens, we use acCRISPR to identify essential genes and call loss-and gain-of-function (LOF and GOF) hits for growth in low pH and high salt culture conditions. We also evaluate the performance of acCRISPR when provided computational predictions of guide activities rather than experimentally determined activities.…”

Section: Introductionmentioning

confidence: 99%

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acCRISPR: An activity-correction method for improving the accuracy of CRISPR screens

Ramesh

Trivedi

Schwartz

et al. 2022

Preprint

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High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of novel and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes, thus determining the fitness effect of disrupted genes. This is accomplished by calculating an optimization metric that quantifies the tradeoff between guide activity and library coverage, which is maximized to accurately classify genes essential to screening conditions. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in gain- and loss-of-function screens under high salt and low pH conditions to identify known and novel genes that were related to stress tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Sgrna Library Cloningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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acCRISPR: An activity-correction method for improving the accuracy of CRISPR screens

Ramesh

Trivedi

Schwartz

et al. 2022

Preprint

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Genome‐wide CRISPR‐Cas9 screen reveals a persistent null‐hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica

Lupish

Hall

Schwartz

et al. 2022

Biotech & Bioengineering

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Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress-induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity.Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome-wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null-hyphal phenotype-ΔRAS2, ΔRHO5, ΔSFL1, ΔSNF2, and ΔPAXIP1. Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L −1 OD −1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway.These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

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Engineering the oleaginous yeast Yarrowia lipolytica to produce nutraceuticals: From metabolic design to industrial applications

Gu,

Lu,

Liu

et al. 2023

Food Bioengineering

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The oleaginous yeast, Yarrowia lipolytica, has garnered much attention and is gaining recognition as a potential industrial platform for nutraceutical production. The superior properties of Y. lipolytica include its transparent inherited background, broad substrate spectrum, and superior environmental tolerance. Specifically, advances in genetic tools and engineering strategies have further widened its applications in metabolic engineering and biomanufacturing. In this review, we summarized developments in synthetic biology and engineering strategies for constructing Y. lipolytica to produce nutraceuticals. We also proposed perspectives on the limitations and challenges in developing microbial Y. lipolytica factories.

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Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

Cited by 33 publications

References 51 publications

acCRISPR: An activity-correction method for improving the accuracy of CRISPR screens

acCRISPR: An activity-correction method for improving the accuracy of CRISPR screens

Genome‐wide CRISPR‐Cas9 screen reveals a persistent null‐hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica

Engineering the oleaginous yeast Yarrowia lipolytica to produce nutraceuticals: From metabolic design to industrial applications

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