Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

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Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽

10.3390/biom10050734 ◽

2020 ◽

Vol 10 (5) ◽

pp. 734 ◽

Cited By ~ 2

Author(s):

Yawei Zhao ◽

Guoquan Li ◽

Yunliang Chen ◽

Yinhua Lu

Keyword(s):

Natural Product ◽

Genome Editing ◽

Genetic Manipulation ◽

Chemical Compounds ◽

Gene Clusters ◽

Future Research ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Research Focus ◽

Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

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Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽

10.1128/msphere.00130-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 68

Author(s):

Kyunghun Min ◽

Yuichi Ichikawa ◽

Carol A. Woolford ◽

Aaron P. Mitchell

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Genetic Analysis ◽

Genome Editing ◽

Drug Targets ◽

Gene Deletion ◽

Genetic Manipulation ◽

Content Type ◽

Biological Features ◽

Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

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Public and Stakeholder Engagement in Developing Human Heritable Genome Editing Policies: What Does it Mean and What Should it Mean?

Frontiers in Political Science ◽

10.3389/fpos.2021.730869 ◽

2021 ◽

Vol 3 ◽

Author(s):

Ana S. Iltis ◽

Sarah Hoover ◽

Kirstin R. W. Matthews

Keyword(s):

Genome Editing ◽

Stakeholder Engagement ◽

Policy Development ◽

Genetic Manipulation ◽

Scientific Practice ◽

Human Embryos ◽

Unrealistic Optimism ◽

Policy Makers ◽

Ethical Concerns ◽

New Treatments

As scientific research pushes the boundaries of knowledge, new discoveries and technologies often raise ethical and social questions. Public responses vary from surprise, to unrealistic optimism about imminent new treatments, confusion, and absolute opposition. Regardless of the intent, the use of a precise gene editing tool on human embryos, such as CRISPR-Cas9, is an example of such a controversial emerging technology. Substantive disagreement about the appropriate research pathways and permissible clinical applications is to be expected. Many ethical concerns, especially related to genetic manipulation of human embryos, are rooted in deeply held moral, religious, or ideological beliefs that science alone cannot address. Today, more scientists and scientific societies as well as policy makers are calling for public and stakeholder engagement in developing guidelines and policies governing scientific practice. We conducted a critical interpretive review of the literature on public and stakeholder engagement in science policy development regarding emerging technologies to determine the ideals that should guide engagement efforts of entities developing recommendations or guidelines on policy for such technologies. We identify and describe five ideals. To illustrate possible applications of these ideals, we review the engagement efforts described in three reports on heritable human genome editing and assess those efforts in light of these ideals. Finally, we recommend possible avenues for engagement that would advance those goals.

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Base editing in Streptomyces with Cas9-deaminase fusions

10.1101/630137 ◽

2019 ◽

Cited By ~ 5

Author(s):

Zhiyu Zhong ◽

Junhong Guo ◽

Liang Deng ◽

Li Chen ◽

Jian Wang ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Genetic Manipulation ◽

Strand Break ◽

High Fidelity ◽

Nucleotide Substitutions ◽

Base Editing ◽

Dna Double Strand Break ◽

Repair Template ◽

Adenine Base

AbstractConventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

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Genome editing for disease resistance in livestock

Emerging Topics in Life Sciences ◽

10.1042/etls20170032 ◽

2017 ◽

Vol 1 (2) ◽

pp. 209-219 ◽

Cited By ~ 2

Author(s):

Chris Proudfoot ◽

Christine Burkard

Keyword(s):

Disease Resistance ◽

Genome Editing ◽

Production Efficiency ◽

Genetic Manipulation ◽

Sequencing Technology ◽

Livestock Industry ◽

Livestock Species ◽

Disease Resistant

One of the major burdens on the livestock industry is loss of animals and decrease in production efficiency due to disease. Advances in sequencing technology and genome-editing techniques provide the unique opportunity to generate animals with improved traits. In this review we discuss the techniques currently applied to genetic manipulation of livestock species and the efforts in making animals disease resistant or resilient.

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Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

Microbial Cell Factories ◽

10.1186/s12934-019-1219-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Wei Shen ◽

Jun Zhang ◽

Binan Geng ◽

Mengyue Qiu ◽

Mimi Hu ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Zymomonas Mobilis ◽

Genome Engineering ◽

Gene Deletion ◽

Genetic Manipulation ◽

Strain Improvement ◽

Genomic Research ◽

Francisella Novicida ◽

A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System

Microorganisms ◽

10.3390/microorganisms8050754 ◽

2020 ◽

Vol 8 (5) ◽

pp. 754

Author(s):

Youran Li ◽

Hanrong Wang ◽

Liang Zhang ◽

Zhongyang Ding ◽

Sha Xu ◽

...

Keyword(s):

Success Rate ◽

Genome Editing ◽

Bacillus Licheniformis ◽

Genetic Manipulation ◽

Inducible Promoter ◽

Bacterial Cells ◽

Protospacer Adjacent Motif ◽

Constitutive Overexpression ◽

Short Palindromic Repeat ◽

Overexpression System

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

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Genetic Modification for Wheat Improvement: From Transgenesis to Genome Editing

BioMed Research International ◽

10.1155/2019/6216304 ◽

2019 ◽

Vol 2019 ◽

pp. 1-18 ◽

Cited By ~ 14

Author(s):

Nikolai Borisjuk ◽

Olena Kishchenko ◽

Serik Eliby ◽

Carly Schramm ◽

Peter Anderson ◽

...

Keyword(s):

Genome Editing ◽

Genetic Manipulation ◽

Loss Of Function ◽

Genetic Determinants ◽

Biotic And Abiotic Stresses ◽

New Techniques ◽

Yield And Quality ◽

Health Promoting ◽

Gene Structure And Expression ◽

Tools And Techniques

To feed the growing human population, global wheat yields should increase to approximately 5 tonnes per ha from the current 3.3 tonnes by 2050. To reach this goal, existing breeding practices must be complemented with new techniques built upon recent gains from wheat genome sequencing, and the accumulated knowledge of genetic determinants underlying the agricultural traits responsible for crop yield and quality. In this review we primarily focus on the tools and techniques available for accessing gene functions which lead to clear phenotypes in wheat. We provide a view of the development of wheat transformation techniques from a historical perspective, and summarize how techniques have been adapted to obtain gain-of-function phenotypes by gene overexpression, loss-of-function phenotypes by expressing antisense RNAs (RNA interference or RNAi), and most recently the manipulation of gene structure and expression using site-specific nucleases, such as CRISPR/Cas9, for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to increase yield, improve nutritional and health-promoting qualities in wheat, and enhance the crop’s resistance to various biotic and abiotic stresses.

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A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

Royal Society Open Science ◽

10.1098/rsos.170095 ◽

2017 ◽

Vol 4 (5) ◽

pp. 170095 ◽

Cited By ~ 115

Author(s):

Tom Beneke ◽

Ross Madden ◽

Laura Makin ◽

Jessica Valli ◽

Jack Sunter ◽

...

Keyword(s):

Genome Editing ◽

High Throughput ◽

Large Scale ◽

Leishmania Major ◽

Fluorescent Protein ◽

Genetic Manipulation ◽

Gene Knockout ◽

Guide Rna ◽

Functional Studies

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

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