Allele-specific genome editing using CRISPR–Cas9 is associated with loss of heterozygosity in diploid yeast

Vries, Arthur R. Gorter de; Couwenberg, Lucas G.F.; Broek, Marcel van den; Cortés, Pilar de la Torre; Horst, Jolanda ter; Pronk, Jack T.; Daran, Jean‐Marc

doi:10.1093/nar/gky1216

“…The types of deletions observed in the uracil- and histidine-auxotrophs are consistent with heterogeneous editing events resulting in loss of heterozygosity 28 – 30 . To extend these observations, we used Nanopore sequencing to better assess the spectrum of large deletions that are often overlooked in Cas9-editing studies.…”

Section: Resultssupporting

confidence: 60%

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

Slattery

¹

,

Wang

²

,

Giguere

³

et al. 2020

Sci Rep

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The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

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“…The types of deletions observed in the uracil-and histidine-auxotrophs are consistent with heterogeneous editing events resulting in loss of heterozygosity [27][28][29] . To extend these observations, we used Nanopore sequencing to better assess the spectrum of large deletions that are often overlooked in Cas9-editing studies.…”

Section: /25supporting

confidence: 59%

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

Slattery

¹

,

Wang

²

,

Kocsis

³

et al. 2020

Preprint

0

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The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ∼2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains. pathways of P. tricornutum and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil-and histidine-requiring phenotypes, respectively. Individual auxotroph strains are characterized by loss of heterozygosity at the edited alleles, and Nanopore sequencing of the edited population reveals large, heterogeneous deletions up to ∼ 2.7 kb. The uracil and histidine auxotrophs and their respective complementation markers are a potential alternative to antibiotic-based selection of plasmids in P. tricornutum. Our results also suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering.

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“…We cannot exclude rare genomic abnormalities (below FISH limit of detection) or a distinct locus genotoxicity. Gorter de Vries et al. (2019) recently warned of the LOH risk not by loss of genetic material but by replacement of the targeted sequence by a copy of the opposite allele.…”

Section: Discussionmentioning

confidence: 99%

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells

Prat

¹

,

Toutain²,

Boutin

³

et al. 2020

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Summary CRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations.

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Allele-specific genome editing using CRISPR–Cas9 is associated with loss of heterozygosity in diploid yeast

Cited by 30 publications

References 50 publications

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells

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