Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

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Abstract

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

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Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

Woong Hwang, Yanfang Fu, Deepak Reyon … (2013)

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.

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Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.

Y Ishino, H Shinagawa, K Makino … (1987)

The iap gene in Escherichia coli is responsible for the isozyme conversion of alkaline phosphatase. We analyzed the 1,664-nucleotide sequence of a chromosomal DNA segment that contained the iap gene and its flanking regions. The predicted iap product contained 345 amino acids with an estimated molecular weight of 37,919. The 24-amino-acid sequence at the amino terminus showed features characteristic of a signal peptide. Two proteins of different sizes were identified by the maxicell method, one corresponding to the Iap protein and the other corresponding to the processed product without the signal peptide. Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid. The Iap protein seems to be associated with the membrane.

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A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action

Kira Makarova, Nick V. Grishin, Svetlana Shabalina … (2006)

Background All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis. Results The protein sequences of the numerous cas gene products were classified into ~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly. Conclusion We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids. This article was reviewed by: Eric Bapteste, Patrick Forterre, and Martijn Huynen. Open peer review Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen. For the full reviews, please go to the Reviewers' comments section.

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Journal

Journal ID (nlm-ta): Genetics

Journal ID (iso-abbrev): Genetics

Journal ID (hwp): genetics

Journal ID (pmc): genetics

Journal ID (publisher-id): genetics

Title: Genetics

Publisher: Genetics Society of America

ISSN (Print): 0016-6731

ISSN (Electronic): 1943-2631

Publication date (Print and electronic): August 2013

Publication date (Print): August 2013

Publication date PMC-release: August 2013

Volume: 194

Issue: 4

Pages: 1029-1035

Affiliations

[* ]Genetics Training Program, University of Wisconsin, Madison, Madison, Wisconsin 53706

[† ]Laboratory of Genetics, University of Wisconsin, Madison, Madison, Wisconsin 53706

[‡ ]Department of Biochemistry, University of Wisconsin, Madison, Madison, Wisconsin 53706

[§ ]Integrated Program in Biochemistry, University of Wisconsin, Madison, Madison, Wisconsin 53706

[†† ]Laboratory of Cell and Molecular Biology, University of Wisconsin, Madison, Madison, Wisconsin 53706

[** ]Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706

Author notes

Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.152710/-/DC1

[1 ]Corresponding authors: 6204B Biochemical Sciences Bldg., 440 Henry Mall, Madison WI 53706. E-mail: mharrison3@ 123456wisc.edu ; 2204B Biochemical Sciences Bldg., 440 Henry Mall, Madison, WI 53706. E-mail: wildonger@ 123456wisc.edu ; 227D Robert M. Bock Labs, 1525 Linden Dr., Madison, WI 53706. E-mail: oconnorgiles@ 123456wisc.edu

Article

Publisher ID: 152710

DOI: 10.1534/genetics.113.152710

PMC ID: 3730909

PubMed ID: 23709638

SO-VID: 288c425f-9749-4fd3-a265-a3c6209dab3d

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Available freely online through the author-supported open access option.

History

Date received : 09 May 2013

Date accepted : 23 May 2013

Page count

Pages: 7

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ScienceOpen disciplines: Genetics

Keywords: crispr rna,cas9,homologous recombination,genome engineering,drosophila

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ScienceOpen disciplines: Genetics

Keywords: crispr rna, cas9, homologous recombination, genome engineering, drosophila

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

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Genome Engineering using CRISPR

Most cited references 18

Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.

A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action

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