We have recently optimized crispr cpf1 renamed cas12a system in. Crispr stands for clustered regularly interspaced short palindromic repeats. Vejnar, miguel morenomateos, daniel cifuentes, ariel a. Crisprcas9directed gene editing for the generation of. As donor dnas we used pcr products and gelpurified restriction fragments of different sizes, all containing exon 6 and in which we had destroyed the crispr target site by the introduction of silent mutations fig. This protocol describes the methods used to generate crispr reagents. Various crisprcas9based methods allow functional genome engineering to manipulate the genome in many organisms including zebrafish. Protocol optimized crispr cas9 system for genome editing in zebrafish charles e. Defining endogenous barcoding sites for crisprcas9based.
This page lists plasmids required to practice crispr casrnaguided nuclease rgn technology in zebrafish. Cas9 mediated genome engineering is transcending biological re. Detection of multiple genome modifications induced by the crisprcas9 system. These are the sources and citations used to research autism, zebrafish, crispr cas9. In addition, cas9 can function with either crrna and tracrrna together or sgrna to efficiently induce targeted alterations in cultured human cells8,9. Pdf background the crisprcas9 system has become a regularly used.
This basic protocol outlines the steps necessary to prepare both plasmids for transfection. Generation of genomic deletions in mammalian cell lines via. Crisprcas9directed genome editing of cultured cells. Dec 15, 2014 next we coinjected linear donor dna together with cas9 mrna and sgrna into alb b4 embryos to test whether hdr can lead to repair of the mutation. Generation of genetically modified mice using the crispr cas9. Protocol for genome editing via the rnaguided cas9 nuclease in zebrafish embryos1 1. Crisprcas9 genome engineering in saccharomyces cerevisiae cells.
In zebrafish, lossoffunction alleles have been successfully produced. Giraldez chapter 10 editing the mouse genome using the crispr cas9 system introduction editing the mouse genome using the crispr cas9 system. Demonstrated protocol crisprcas9 genome editing in stem. Targeted mutagenesis in atlantic salmon salmo salar l. Genetics, issue 8, danio rerio, zebrafish, crisprcas9, embryo. Zebrafish embryo microinjection ribonucleoprotein delivery using the altr crispr cas9 system contributed by jeffrey essner, phd, associate professor department of genetics, development, and cell biology, iowa state university, ames, ia, usa the methods presented here are provided by a customer who has used the altr crispr cas9 system. Crisprcas9based genome engineering of zebrafish using a. In vitro synthesis of capped cas9 mrna the full length of humanized cas9 cdnas with double nls were cloned into pxt7 vector ampresistant and linearized by xbai neb.
In the zebrafish community, knockout reports are constantly increasing but insertion studies have been so far more challenging. Crispr cas9 has been used for a wide range of experimental applications, and zebrafish has been a key model organism to test and validate strategies for genome editing 1,2. Flavell4,5,7 1institute for immunology, perelman school of medicine, university ofpennsylvania, philadelphia, pennsylvania. Church1 1department of genetics, harvard medical school, boston, massachusetts. Genomewide crispr cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genomewide scale and studying the resulting phenotypic alterations. The best studied crispr cas9 system is from the bacteria streptococcus pyogenes and this is the system that has been used for most zebrafish genome engineering work. Pdf the zebrafish danio rerio has emerged in recent years as a powerful vertebrate. These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms. A rapid crisprcasbased mutagenesis assay in zebrafish for. Efficient production and identification of crisprcas9generated. Repair of crispr generated doublestranded breaks dsbs by nonhomologous end joining nhej leads to insertions and deletions indels which may result in loss of function of the.
Here we present a protocol for the conversion of egfptransgenic zebrafish lines into lines expressing gal4 from the same locus. Oct 24, 20 genome engineering using the crispr cas9 system. Design 20nt target sequence crispr targets can be designed using various online tools e. Crispr cas systems, in particular the type ii systems containing the cas9 endonuclease, have been exploited for their ability to bind desired nucleic acid sequences, as well as direct sequencespeci. Before explaining how the crispr cas9 method can be used for prokaryote genome editing, the inheritable prokaryotic immune system has to be outlined as the crispr technique is based upon this natural phenomena jinek et al. Crispr methods and protocols magnus lundgren springer. Optimized crisprcas9 system for genome editing in zebrafish. Yang,1,2,3 susan byrne,1,3 joshua pan,2 and george m. Congress also may have a role to play with respect to regulation, research and development, and economic competitiveness associated with crispr cas9 gene editing and future biotechnology products. Pdf genome editing in zebrafish using highfidelity cas9. Crisprcas9mediated conversion of egfp nature protocols. Crispr gene editing methods and protocols yonglun luo.
Zebrafish genome engineering using the crisprcas9 system. Those repeats are elements of the genetic code in dna. Until recently, functional studies in salmonids appeared to be difficult. Analysis of single nucleotide variants in crisprcas9. Crisprcas9 offers unprecedented possibilities for genomic manipulation that can be. Part of the research and perspectives in neurosciences book series. Because single guide rnas sgrnas have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool crisprscan to design highly efficient sgrnas. Costefficient and effective mutagenesis in zebrafish with crispr. Genetics, issue 8, danio rerio, zebrafish, crisprcas9. Crispr dna vector n nucleus n u c l e a r l o c al iz t on t r a n s c r i p t i o n c a s 9 target r n p c o m p l e x cas9 cas9 cas9 cas9 cleavage maximum efficiency, minimal offtarget cleavage with crisprcas9 rnp complex by transfecting cas9 protein together with the targeting grna, we achieve higher cutting efficiencies and lower off. The nextgeneration sequencing identifies a growing number of candidate genes associated with human genetic diseases, which inevitably requires efficient methods to validate the causal links between genotype and phenotype. The recently developed crispr cas9 based barcoding method is, although highly promising, difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome. Crispr cas9 rnp deliveryzebrafish embryosj essner 119 kb crispr cas9 rnp. We have recently optimized crispr cpf1 renamed cas12a system in zebrafish.
With help from our collaborators, we also make usersubmitted methods available for genome editing in other model systems. Crisprcas9mediated knockin and knockout in zebrafish. The presence of novel heteroduplex dna indicates that indels were generated by the crisprinjection. As crispr clustered regularly interspaced short palindromic repeats cas9 technology becomes more mainstream in life science research, it becomes critical for undergraduate instructors to devise engaging ways to bring the technology into their classrooms. The proposed combination of efficient mutagenesis protocols, rapid noninvasive phenotyping and sensitive genotyping holds great potential to. Reduction of thiolmodified oligonucleotides 575 kb reduction of thiolmodified oligonucleotides 575 kb resuspension of duplexed oligonucleotides. Benjamin feldman from nichd for technical advice on microinjections. The introduction of engineered sitespecific dna endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes f0, and then. Cas9 enzymes together with crispr sequences form the basis of a technology known as crispr cas9 that can be used to edit genes within organisms. Here, we took advantage of the crispr cas9 technique to insert an. This unit describes a detailed protocol for performing an f 0 screen using the crispr cas9 system in zebrafish starting. Crispr cas9 is a robust system to produce disruption of genes and genetic elements. Pdf crisprcas9mediated knockin and knockout in zebrafish.
Efficient production and identification of crisprcas9. We describe here protocols for creating insertion or deletion indel mutations or precise sequence modifications in zebrafish genes using customizable crispr cas9 rnaguided nucleases rgns. This protocol describes how to generate and genotype mutants using an optimized crisprcas9 genomeediting system in zebrafish crisprscan. The zebrafish danio rerio has emerged in recent years as a. Genome engineering via crispr cas9 systems presents a compilation of chapters from eminent scientists from across the globe who have established expertise in working with crispr cas9 systems.
To circumvent this limitation, we developed a simple and versatile protocol to achieve. A crispr cas9 vector system for tissuespecific gene disruption in zebrafish. Zebrafish danio rerio has been one of the vertebrate model species where crispr cas9 has been applied very extensively for. Protocol for preparation of spcas9 mrna for microinjection. New developments in crisprcasbased functional genomics. Currently, targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative simplicity to which they can. Positive control crispr cas9 protein editing in hek293ft cells. To help meet this challenge, the national science foundation sponsored a workshop for undergraduate instructors in june. Crisprcas9 mutagenesis protocol for zebrafish cloningfree. Pdf a simplified method for identifying early crisprinduced. Genome editing using crispr cas9 has advanced very rapidly in its scope, versatility and ease of use. Dna integration results from cotransforming 1 a single plasmid pcas that coexpresses the cas9 endonuclease and a uniquely engineered single guide rna sgrna.
The following crispr plasmids have been designed for use in zebrafish. Allowseveralweeks2k3weeksusuallyforeachsinglecelltogrowintoavisiblecolony, inspectcellseveryweekandchange. Origene also designed a set of donor cassettes for construction of donor vectors. A guide for the laboratory use of zebrafish danio rerio. Protocols for crispr genome editing in your model system. Crispr cas9 protein for genome editing getting started guide. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using crispr cas9. The best studied crispr cas9 system is from the bacteria s. Cas9based genome editing in zebrafish washington university. Genome engineering using the crisprcas9 system nature. User guides and protocols integrated dna technologies. The presence of novel heteroduplex dna indicates that indels were.
Genome editing using crisprcas9based knockin approaches in. This volume presents a list of cuttingedge protocols for the study of crispr cas defense systems and their applications at the genomic, genetic, biochemical and structural levels. Crisprcas9 is a geneediting technology which involves two essential components. Genome engineering via crisprcas9 system 1st edition. These include luciferaseloxppuroloxp, tgfploxppuroloxp and trfploxpbsdloxp. This protocol describes the methods used to generate crispr reagents for. Inject 10 nl of grnacas9 mixture into animal side at 1 cell stage. We now demonstrate that crispr cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86. This book includes guidelines to select the best crispr cas system and target sites, covering crispr grna design, crispr delivery and activity quantification, and examples of applying crispr gene editing in human pluripotent stem cells.
In this study, we characterized 78 highquality endogenous sites in the zebrafish genome that can be used as crispr cas9 based barcoding sites. Introduction editing endogenous genomic dna in eukaryotic cells is now possible with crispr cas9 technology. Materials cas9 plasmid addgene, plasmid id 41815 as bacterial stab in agar. To test the efficiency of crisprcas9mediated genome editing in mouse models using either cas9 recombinant protein, or cas9 mrna and in vitrotranscribed short guide rna sgrna, we perform pronuclear injections and culture the zygotes to the blastocyst stage. This protocol describes the use of a highly efficient kit to synthesize sgrnas using dna oligonucleotides oligos, similar to other approaches that have been previously described 32. Precise editing of the zebrafish genome made simple. The crisprcas9 system has been successfully applied in zebrafish, including knockout, highthroughput mutagenesis screens, multiplex knockout, conditional knockout, and knockin.
Targeted mutagenesis in zebrafish using crispr rnaguided. The steps described herein provide a simple, robust, and economical crispr cas9 strategy to engineer zebrafish knockout lines. This crispr cas9 protocol describes how to use t7 endonuclease i t7e1 to detect ontarget crispr cas9 editing events in cells. Fully functional crispr cas enzymes will introduce a doublestrand break dsb at a specific location based on a grnadefined target sequence. Here, we took advantage of the crisprcas9 technique to insert an. The crispr cas9 system is a diverse family of endonucleases found in archaeal and bacterial genomes that have been adapted for use in engineering metazoan genomes 1,2. Giraldez1,2,5 1department of genetics, yale university school of medicine, new haven, connecticut 06510.
Microinjection in zebrafish for genome editing and. Efficient genome editing in zebrafish using a crisprcas system. Middle entry clone for gateway containing a zebrafish codonoptimized cas9 flanked by 2. Introduction the guideit crispr cas9 system green, cat. A novel technique based on in vitro oocyte injection to improve crispr cas9 gene editing in zebrafish skip to main content thank you for visiting. The crispr see glossary cas9 system is a diverse family of endonucleases found in archaeal and bacterial genomes that have been adapted for use in engineering metazoan genomes 1, 2. Crispr cas9 directed gene editing for the generation of lossoffunction mutants in highthroughput zebrafish f 0 screens. The crispr cas9 system biotechnological impact has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient crispr based endonucleases. This work was supported by the intramural research program of the national human genome research institute ziahg20038605. This bibliography was generated on cite this for me on thursday, august 27, 2015. Protocol 1 generation of genetically modified mice using the crisprcas9 genomeediting system jorge henaomejia,1,2,6,7 adam williams,3,6,7 anthony rongvaux,4 judith stein,4,5 cynthia hughes,4,5 and richard a.
With its variety of applications, the crispr cas9 genome editing technology has been rapidly evolving in the last few years. This protocol describes a method for crisprcas9mediated genome editing that results in scarless and markerfree integrations of dna into saccharomyces cerevisiae genomes. Moreover, this novel seamless geneediting strategy in zebrafish provides a valuable new transgenesis technique. Zebrafish were cared for as described in the zebrafish book. A novel technique based on in vitro oocyte injection to. Varshney and other members of the burgess laboratory and nhgri zebrafish core for improving the protocols. As a result, there is not yet an established procedure for the routine. The crispr cas9 system requires only the redesign of the crrna to change target specificity.
Demonstrated protocol crispr cas9 microinjection e mbryo microinjection with crispr cas9 in mice and zebrafish mouse to test the efficiency of crispr cas9 mediated genome editing in mouse models using either cas9 recombinant protein, or cas9 mrna and in vitrotranscribed short guide rna sgrna, we perform pronuclear. Recently, zebrafish, with sufficiently highthroughput capabilities, has become a favored option to study human pathogenesis. A recent exciting development is the use of the dcas9 version of the crispr cas9 system to target protein domains for transcriptional regulation 26, 51, 52, epigenetic modification 25, and microscopic visualization of specific genome loci 27. The sgrna was then resuspended in depc water and stored at 80c. Dec 16, 2015 the introduction of new genome editing tools such as zfns, talens and, more recently, the crisprcas9 system, has greatly expanded the ability to knockout genes in different animal models, including zebrafish.
Jul 25, 2017 pcr and sequencing analysis showed that 6 out of 16 f0 injected fish. Mit crispr tool, chopchop, casblastr and many others. Demonstrated protocol crisprcas9 genome editing in stem cells e mbryo microinjection with crisprcas9 in mice and zebrafish mouse to test the efficiency of crisprcas9mediated genome editing in mouse models using either cas9 recombinant protein, or cas9 mrna and in vitrotranscribed short guide rna sgrna, we perform pronuclear. Idt scientists have developed detailed lipofection and electroporation protocols for using the altr crispr cas9 system and the altr crispr cpf1 system in mammalian cells table 1. Introduction of crispr cas9 methods clustered regularly interspaced short palindromic repeats, crispr associated protein 9 have led to a huge surge in the use of precision genome editing for. Crispr cas9 based approaches are currently used for multiple geneediting purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Understanding the biological function behind key proteins is of great concern in atlantic salmon, both due to a high commercial importance and an interesting life history. Optimized crisprcpf1 system for genome editing in zebrafish. To test the efficiency of crispr cas9 mediated genome editing in mouse models using either cas9 recombinant protein, or cas9 mrna and in vitrotranscribed short guide rna sgrna, we perform pronuclear injections and culture the zygotes to the blastocyst stage. Synthetic crisprcas9 reagents facilitate genome editing.
Crispr cas9 system for plant genome editing within the. This conversion allows the indepth analysis of the former egfp. This second edition details new emerging areas of zebrafish research focusing on genetics and genomics. Crisprcas9 mutagenesis protocol for zebrafish cloning. As crispr cas9 genome editing has begun to revolutionize molecular biology research, the zebrafish community has responded in stride by developing crispr cas9 techniques for the zebrafish as well. Crisprcas9 and gal4uas combination for cellspecific gene. Precise and efficient genome editing in zebrafish using. A rapid and cheap methodology for crisprcas9 zebrafish. Programmable base editing of zebrafish genome using a. Cas9 or crispr associated protein 9 is an enzyme that uses crispr sequences as a guide to recognize and cleave specific strands of dna that are complementary to the crispr sequence. Methods and protocols guides readers through techniques that have been developed specifically for the analysis. Crisprinduced indels can be detected in the late zebrafish.
Cas9 alone pspcas9 or cas9 with an invariant sgrna scaffold and cloning sites for. Featuring contributions from leading figures in the crispr cas field, this laboratory manual presents a stateoftheart guide to the technology. Crispr handbook enabling genome editing and transforming life science research. Protocol 1 preparation of cas9 and grna plasmids plasmids containing cas9 and the guide rna are necessary for cas9 mediated genome editing. Inject 10 nl of grna cas9 mixture into animal side at 1 cell stage. Programmable base editing of zebrafish genome using a modified crisprcas9. Protocol for genome editing via the rnaguided cas9 nuclease in. The use of base editing enables precise genetic modifications in model animals.
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