Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9

Iwata, Satoru; Yoshina, Sawako; Suehiro, Yutaka; Hori, Shigeru; Mitani, Shohei

doi:10.1038/srep33840

Cited by 33 publications

(55 citation statements)

References 29 publications

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“…Two DSBs have been used to make chromosomal rearrangements, including inversions translocations, and large deletions (Blasco et al 2014;Chen et al 2014;Choi and Meyerson 2014;Ghezraoui et al 2014;Maddalo et al 2014;Paix et al 2014;Iwata et al 2016;Dejima et al 2018). We found that adding a second DSB at a distance of 340 bp from the initial DSB ( Figures 5A and S2) This double DSB strategy also worked well using a variety of linear and circular doublestranded repair templates with 500 bp homology arms to insert fluorescent reporters: 21% dpy-10 mutants had precise insertions of 8221 bp, 17% had precise insertions of 5060 bp, and 36% had precise insertions of 2138 bp ( Figure 5B).…”

Section: Efficient Strategies To Insert Large Dna Fragments At Wide-rmentioning

confidence: 99%

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Farboud

Severson

Meyer

2018

Preprint

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The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes, both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We devised strategies to insert long (10 kb) exogenous sequences or incorporate multiple nucleotide substitutions at considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex-dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. Our studies revealed a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9 targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

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Section: Efficient Strategies To Insert Large Dna Fragments At Wide-rmentioning

confidence: 99%

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Farboud

Severson

Meyer

2018

Preprint

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“…GRIBCG is a fast and easy-to-use tool for the selection of sgRNAs in the rational design of balancer chromosomes. While previous work has demonstrated successful generation of balanced regions in C. elegans and Danio rerio [5,7], our tool is the first designed to create a completely balanced chromosome with the use of a single sgRNA. Experimentally, using a single sgRNA would eliminate the need for multiple rounds of transformation, and decrease the number of generations that need to be screened in order to identify a completely balanced chromosome.…”

Section: Resultsmentioning

confidence: 99%

“…In plant breeding, balancer chromosomes could help preserve the advantages of heterosis without full apomixis [4]. CRISPR/Cas9 genome editing can generate inverted regions similar to the rearrangements present in balancer chromosomes ( Figure 1) [5,6]. Chromosomal rearrangements have been reported in C. elegans and zebrafish germlines, and in pig, mouse, and human somatic cells [5,[7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

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GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

Merritt

Cheung

2018

Preprint

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BackgroundBalancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research. ResultsHere, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes. ConclusionGRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

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“…Examples of using CRISPR/Cas9 to engineer chromosome rearrangements in other model systems include the creation of an oncogenic translocation in mice (5), translocations in yeast (6), translocations and inversions in C. elegans (7,8), and inversions in zebrafish (9). This study represents the first report of a chromosomal rearrangement induced by CRISPR/Cas9 in the Drosophila germline.…”

Section: Introductionmentioning

confidence: 91%

A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila

Reed

2019

Preprint

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Using CRISPR/Cas9 to simultaneously induce mutations in two or more target genes, commonly referred to as multiplexing, may result in chromosomal rearrangements such as inversions or translocations. While this may be undesirable in some contexts, the ability to recover chromosomal rearrangements targeted to specific sites in the genome is potentially a powerful tool. Before developing such tools, however, it is first important to measure the frequency with which chromosome rearrangements are induced by CRISPR/Cas9 multiplexing. To do this, we have developed a selfselecting screening system using a Drosophila line that carries an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of this autosynaptic stock are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, and if it is analogous to the inversion in the male autosynaptic line, then it is possible to recover progeny in which aneuploidy is reduced and viability is restored. Using this selfselection method, we screened 130 females and recovered one new autosynaptic element. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of a breakpoint induced precisely between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. Also, in using this particular system, the recovery of chromosomal rearrangements was not a high frequency event.

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Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9

Cited by 33 publications

References 29 publications

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Strategies for Efficient Genome Editing Using CRISPR-Cas9

GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila

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