Plasmid-based CRISPR-Cas9 gene editing in multiple<i>Candida</i>species

Lombardi, Lisa; Oliveira-Pacheco, João; Butler, Geraldine

doi:10.1101/557926

Cited by 2 publications

(4 citation statements)

References 45 publications

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“…This means that the DSBs induced by the CRISPR–Cas9 system are lethal, but can be repaired at low frequency by nonhomologous end joining in C. tropicalis . A similar result, in which only a single base near the cut site was deleted, was recently obtained with a replicating plasmid‐based CRISPR–Cas9 system (Lombardi et al, ). Nonhomologous end joining of DSBs is expected to happen more frequently with plasmid‐based systems.…”

Section: Resultssupporting

confidence: 68%

“…We tried to use the tRNA Ala gene from C. albicans to deliver sgRNA in C. tropicalis , but failed. Recent research has shown that the tRNA Ala gene of Candida parapsilosis can be function in C. tropicalis (Lombardi et al, ). Perhaps this gene might help improve the multi‐gene disruption efficiency of our CRISPR–Cas9 system.…”

Section: Discussionmentioning

confidence: 99%

“…Another advantage of the system is that the operation is simple and quick when editing another locus, because only the 20 bp target sequence is unique in the system. As we prepared this manuscript, Lombardi, Oliveira‐Pacheco, and Butler () described a plasmid‐based CRISPR–Cas9 system for C. tropicalis that displayed high disruption efficiency for the ADE2 gene. The CRISPR–Cas9 system has also been reported to facilitate cell factory development and synthetic genomes in Saccharomyces cerevisiae (Jakociunas et al, ; Shao et al, ; J. T. Zhou, Wu, Xue, & Qin, ).…”

Section: Introductionmentioning

confidence: 99%

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A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Zhang

Liu

et al. 2019

Biotech & Bioengineering

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Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time‐consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β‐carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

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Section: Resultssupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Zhang

Liu

et al. 2019

Biotech & Bioengineering

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“…486 487 Tagging Cse4. C. parapsilosis strains CLIB214 and 90-137 were edited using a tRNA 488 plasmid based CRISPR-Cas9 gene editing system as described byLombardi et al 489 (Lombardi et al 2017, 2019a. Primers gRNA_CSE4_TOP and gRNA_CSE4_BOT were 490annealed and cloned into pCP-tRNA and 5 µg plasmid was transformed together with 5 µg 491 of a 594 bp synthetic DNA fragment containing a section of the H3 histone variant Cse4 with 492 a 3xHA (hemagglutinin) tag inserted between amino acids 69 and 70, and 250bp homology 493 arms (Integrated DNA Technologies USA, Supplemental_Fig._S1).…”

mentioning

confidence: 99%