Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae

Hao, Huanhuan; Wang, Xiaofei; Jia, Haiyan; Yu, Miao; Zhang, Xiaoyu; Tang, Hui; Zhang, Liping

doi:10.1016/j.ab.2016.07.008

Cited by 27 publications

(26 citation statements)

References 22 publications

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“…The ability to target a region with multiple sgRNAs has been shown to be beneficial in creating knockout mutations and large deletions in other plant systems, including soybean (Cai et al, ), rice (Srivastava, Underwood, & Zhao, ; Wang et al, ; Zhou, Liu, Weeks, Spalding, & Yang, ), tomato (Brooks, Nekrasov, Lippman, & Eck, ), Arabidopsis thaliana (Gao, Chen, Dai, Zhang, & Zhao, ; Ordon et al, ), and Nicotiana benthamiana (Ordon et al, ), as well as in human cells (He et al, ), zebrafish (Xiao et al, ), and yeast (Hao et al, ). Protospacer sequences have variable probabilities of producing out‐of‐frame mutations, depending on the surrounding microhomology in the genomic DNA available for microhomology‐mediated end‐joining repair (Bae, Kweon, Kim, & Kim, ).…”

Section: Resultsmentioning

confidence: 99%

“…Multiplexing is not solely limited to different genes, as multiple sgRNAs can be used to target a single region. This has been shown to be successful in creating gene deletions as well as large‐scale chromosomal deletions (Cai et al, ; Hao et al, ; He et al, ; Mali et al, ; Xiao et al, ). In our case, we tested the ability to create easy‐to‐detect knockout mutations using two sgRNAs separated by ~200 and ~500 base pairs in protein‐coding genes.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

et al. 2019

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CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR‐Cas9‐mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

et al. 2019

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“…Multiplexing is not solely limited to different genes, as multiple sgRNAs can be used to target a single region. This has been shown to be successful in creating gene deletions as well as large-scale chromosomal deletions (Cai et al, 2018;Hao et al, 2016;He et al, 2015;Mali et al, 2013;Xiao et al, 2013). In our case, we tested the ability to create easy-to-detect knockout mutations using two sgRNAs separated by ~200 and ~500 base pairs in protein-coding genes.…”

Section: Discussionmentioning

confidence: 99%

Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

Mallett

Chang

Cheng

et al. 2019

Preprint

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CRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR-Cas9-mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a "stop codon cassette" with downstream near-effortless genotyping. K E Y W O R D S CRISPR, genome editing, homology-directed repair, multiplexing, Physcomitrella patens, vector system S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Mallett DR, Chang M, Cheng X, Bezanilla M. Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. Plant Direct. 2019;3:1-15.

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“…Larger chromosomal deletions (>30 kb) and chromosome fusion have been achieved in S. cerevisiae through NHEJ repair between endpoints of the DNA breaks, which can facilitate the study of gene clusters or minimal genome research, as well as the study of replication, recombination, and segregation [70,71,72]. In principle, this may open up the possibility to generate CRISPR-Cas9-induced genome rearrangements in C. albicans, in which specific whole-chromosome aneuploidies have been associated with drug resistance and/or cross-adaptation to different drugs [73].…”

Section: Perspectives and Future Directionsmentioning

confidence: 99%

“…Study of the correlation existing between large chromosomal rearrangements and virulence and drug resistance (e.g., aneuploidy and LOH in C. albicans, aneuploidy in C. neoformans) [70][71][72] Directed evolution through CRISPRguided DNA polymerase…”

Section: S Cerevisiaementioning

confidence: 99%

The CRISPR toolbox in medical mycology: State of the art and perspectives

2020

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Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

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Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae

Cited by 27 publications

References 22 publications

Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

The CRISPR toolbox in medical mycology: State of the art and perspectives

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