News & Results

Visit the flyCRISPR Discussion Group for ongoing discussions. We'll also be posting new data and reagents here as they become available.

 

June 10, 2015

Tools for scarless HDR with screenable markers

HDR with dsDNA donors offers mutliple advantages, including the ability to edit over large regions, insert large exogneous sequences, and incorporate selection markers. While selectable markers greatly facilitate identification of engineered organisms, most methods for removing marker cassettes leave behind viral or yeast DNA sequences of 30-90 bp (scars) that can disrupt gene expression. We have generated the collection of cassettes and vectors for rapid cloning of donor vectors for scarless genome engineering that are now available (or in some cases, will be very soon) through the Drosophila Genomics Resource Center. We'll deposit more as we make them, and welcome your feedback.

 

January 8, 2015

Diverse applications of CRISPR-Cas9 Genome Engingeering Workshop, 56th Drosophila Research Conference

We are organizing a workshop on technical advances and application of the CRISPR-Cas9 system for the upcoming Drosophila Research Conference (March 4-8 in Chicago, IL). If you have made advances in the use of this new technology and would like to participate by presenting a 15-minute talk about your CRISPR-Cas9 experiements, please senda brief  description of your project and data to Jill Wildonger (wildonger@wisc.edu), Kate O'Connor-Giles (oconnorgiles@wisc.edu) or Melissa Harrison (mharrison3@wisc.edu).

 

November 22, 2014

Replacing endogenous loci with attP docking sites

Here are are the results of some recent experiments to replace endogenous loci with an attP docking site. All of the experiments were conducted in Vasa-Cas9 flies by injection of two plasmids encoding flanking gRNAs and the pHD-DsRed-attP donor vector. Using this approach, we replaced genes ranging in size from approximately 2 to more than 25 Kb. Interestingly, the efficiency of HDR does not appear to correlate with the size of the locus being deleted. Note that the 28-Kb gene replacement we didn't recover appears to be due to the locus rather than the size of the deletion as we recently tried and failed to replace a smaller subset of the same locus.

 

 

Posted by Kate OCG.

 

September 27, 2014

More updates to Target Finder webtool

Target Finder has been updated to provide the oligonucleotide sequences to order for generating gRNAs using the pU6-BbsI-chiRNA plasmid. Just select target site(s) and click the Design Experiment button in the upper right hand corner of the results page. The overlapping oligo sequences will be displayed.

September 22, 2014

Updates to Target Finder webtool

Target Finder has been updated with the latest version (release 6) of the Drosophila melanogaster genome and additional species have been added. Here's the current list of species Target Finder searches:

Drosophila melanogaster (r_5/dm3)
Drosophila melanogaster (r_6)
D. simulans (annotation DsimV2)
D. simulans (DroSim1)
D. yakuba (DroYak2)
D. mauritiana (Dmau_MS17)
D. sechellia (DroSec1)
D. ananassae (dana_r1.3_FB2011_07)
D. erecta (dere_r1.3_FB2011_08)
D. persimilis (dper_r1.3_FB2010_02)
D. pseudoobscura (dpse_r3.1_FB2013_03)
D. virilis (DroVir3)
D. mojavensis (dmoj_r1.3_FB2011_05)
D. willistoni (dwil_r1.3_FB2010_02)
D. grimshawi (dgri_r1.3_FB2010_02)
Anopheles gambiae (AgamM1)
Anopheles gambiae (AgamS1)
Aedes aegypti (AaegL1)
Apis mellifera (apiMel3)
Tribolium castaneum (TriCas2)
T. castaneum (T cas 4.0 draft)
Caenorhabditis elegans (ce10)

Target Finder also now allows you to select whether you would like your gRNA target sequence to be 20, 19, 18, 17 or 16-nt. The Joung lab recently demonstrated that reducing the gRNA target size can increase specificty while maintaining efficiency.

 

May 10, 2014

New vasa-Cas9 stock now available at the Bloomington Drosophila Stock Center

We have deposited an X-chromosome vasa-Cas9 with the RFP marker in the attP landing site removed to facilitate use with DsRed-marked HDR donors:

55821     y[1] M{vas-Cas9.RFP-]ZH2A w[1118]/FM7a, P{w[+mC]=Tb[1]}FM7-A

The key difference between this line and 51323 is the (-) after the RFP indicating the removal of the landing site marker. Here's a link to all the CRISPR stocks available at Bloomington.

 

An update on HDR donor vectors at Addgene

The pHD-DsRed-attP vector described in Gratz, Ukken et al. (2014) is available through Addgene. The name is slightly different (pDSRed-attP), but it is the same vector. Our apologies for any confusion. The non-attP version, pHD-DsRed, is also at Addgene.

 

March 24, 2014

Join us at the CRISPR Workshop at the 55th Annual Drosophila Research Conference in San Diego this week!

Friday, March 28 1:45-3:45 in the Pacific Ballroom Salon 1

Organizers: Melissa Harrison, University of Wisconsin, Madison; Kate O'Connor-Giles, University of Wisconsin, Madison; and Jill Wildonger, University of Wisconsin, Madison

The fantastic lineup of speakers:

Scott Gratz (O’Connor-Giles Lab, University of Wisconsin Madison). CRISPR and the promise of genome engineering on demand

Fillip Port (Bullock Lab, MRC Cambridge). CRISPR fly design: An optimized toolbox for Drosophila genome engineering

Guanjan Gao (Tsinghua University). Transgenic Cas9/gRNA system mediates high efficient gene targeting in Drosophila

Andrew Bassett (Liu Lab, University of Oxford). Genetic knockouts and homologous targeting in Drosophila cell lines with CRISPR/Cas9 – new tools to investigate gene function.

Luis Alberto Baena (Vincent Lab, MRC National Institute for Medical Research). Different cocktails for various flavours of genomic engineering.

Kelly Beumer (Carroll Lab, University of Utah). Designer mutations with nucleases: Homologous recombination tips and tricks.

The speakers and organizers will participate in a thirty-minute panel discussion following the talks.

Looking forward to seeing you there!

 

February 11, 2014

Cloning vectors for homology-directed repair with positvely marked dsDNA donors now available at Addgene. We've also posted detailed protocols for homology-directed repair with the CRISPR system.

CRISPR Optimal Target Finder now searches a number of genomes including D. simulans, D. yakuba, D. sechellia, D. virilis, Apis mellifera, Tribolium castaneum, two strains of Anopheles gambiae, Aedes aegypti, and Caenorhabditis elegans. We've also improved the visual representation of identified CRISPR locations within inputted sequence.

 

November 5, 2013

New CRISPR Optimal Target Finder tool

Dustin Rubinstein, a postdoctoral researcher in the O’Connor-Giles lab, has teamed up with Ed O’Connor-Giles to build a web tool that identifies CRISPR target sites within a genomic region of interest and evaluates their potential for off-target cleavage using transparent algorithms based on empirical studies. Give it a try and email Dustin with any suggestions or feedback.

 

September 17, 2013

Vasa-Cas9 and U6-tracrRNA flies now available at the Bloomington Drosophila Stock Center

Our flies can now be ordered from the Bloomington Drosophila Stock Center through this link. They are still in the process of getting all the stock information up on their site so you can't find them by searching yet. Until then, here's all the information you should need to order them:

 

51321    w[1118]; PBac{y[+mDint2] w[GMR.PHb]=U6-tracrRNA}VK00037/CyO, P{Wee-P.ph0}2

51322    y[1] M{w[GMR.PHb]=U6-tracrRNA}ZH-2A w[1118]/FM7c, P{w[+mC]=GAL4-Kr.C}DC1, P{w[+mC]=UAS-GFP.S65T}DC5, sn[+]

51323    y[1] M{vas-Cas9}ZH-2A w[1118]/FM7c

51324    w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00027

51325    w[1118]; PBac{y[+mDint2]=vas-Cas9,U6-tracrRNA}VK00027

51326    y[1] M{vas-Cas9,U6-tracrRNA}ZH-2A w[1118]

 

These are unpublished reagents. If you publish with them, please acknowledge Kate O'Connor-Giles, Jill Wildonger and Melissa Harrison or cite flyCRISPR.molbio.wisc.edu.

 

July 18, 2013

Cas9, tracrRNA flies

Germline expression of Cas9 may improve CRISPR RNA/Cas9 targeting efficiency, so we’ve generated flies that express Cas9 under the control of the vasa promoter. In addition, we’ve generated flies and reagents for separately expressing the sequence-specific CRISPR (cr) and Cas9-interacting trans-activating CRISPR (tracr) RNAs. This dual RNA system will limit the injections to only the sequence-specific crRNA and may increase cleavage efficacy above that achieved with chimeric guide RNAs. Specifically, we’ve generated flies that express tracrRNA under the control of the snRNA:U6:96Ab promoter with and without Cas9. A construct for cloning crRNAs for use with these flies will be available through Addgene.

The lines below have been deposited at the Bloomington Drosophila Stock Center. They’re currently being expanded and should be available for distribution in about a month. We’ll post a link here as soon as they’re available.

1. vasa>Cas9 inserted into the ZH-2A attP site on the X (lane 2 below)

2. vasa>Cas9 inserted into the VK27 attP site on 3 (lane 6)

3. U6>tracrRNA inserted into ZH-2A on X

4. U6>tracrRNA inserted into the VK37 attP site on 2

5. vasa>Cas9, U6>tracrRNA inserted into ZH-2A on X (lane 4)

6. vasa>Cas9, U6>tracrRNA inserted into VK27 on 3 (lane 8)

Two new papers show high-efficiency cleavage in Drosophila with injection of CRISPR components as RNAs

Bassett et al. and Yu et al. both show highly efficient cleavage of several different target sites when they inject Cas9 and chiRNAs as in vitro transcribed RNAs. Using single chiRNAs to generate indels via NHEJ, the two labs tested a variety of targets and saw germline transmission efficiencies ranging from 0-100%. This is compared to the 6-27% that we’ve observed for generating similar modifications via plasmid DNA injections targeting two different genes, yellow and rosy (Gratz et al., 2013 and below). While there are clearly significant locus and target specific differences that make direct comparison difficult, RNA injections may be more efficient than DNA due to differences in concentration and/or timing of expression relative to germline development. So far, a side-by-side comparison has not been completed. Increased efficiency will greatly facilitate molecular screening. However, a high rate of cleavage, if biallelic, may prove problematic when targeting essential genes. It will also be of interest to determine the rate of off-target effects with different approaches. Encouragingly, neither our groups or Bassett et al., who employed high-resolution melt analysis to probe many different loci, have seen any evidence of off-target cleavage.

 

July 10, 2013

Generation of targeted mutations in rosy

We used three chiRNAs, R1, R2 and R3 to generate independent rosy mutations via non-homologous end joining (NHEJ; Figure 1 and Table 2). Sequence alignments reveal cleavage and imprecise repair at predicted cut sites (Figure 2). One R3-guided event resulted in a larger indel that is not shown. 8-27% of injected flies yielded mutant progeny (Table 1), which is significantly higher efficiency than we observed in our experiments targeting yellow (Gratz et al., 2013) suggesting significant locus and target-specific differences.

We also targeted the rosy locus for replacement with an attP recombination docking site by co-injecting two chiRNAs, R5′ targeting the 5′ end of rosy and R3′ targeting the 3′ end (Figure 1), along with a single-stranded oligodeoxynucleotide (ssODN) repair template that includes a 50-nt attP site flanked by 60-nt homology arms corresponding to sequences 5′ and 3′ of rosy. In 18 crosses, we did not recover the attP replacement of rosy, but did recover the precise deletion of the 6.1-kb rosy locus without attP incorporation from 1/18 crosses (5.6%).

 

Figure 1. chiRNAs used to target rosy. chiRNAs were injected as a DNA plasmid for in vivo transcription under control of the Drosophila snRNA:U6:96Ab promoter for in vivo transcription.

 

Figure 2. Sequences of mutations in rosy using R1, R2 or R3 chiRNA to target Cas9.

 

Table 1. Germline transmission rates of targeted mutations in rosy. Flies injected with plasmids for expressing Cas9 and the indicated chiRNAs with or without an ssODN template were outcrossed and progeny screened for rosy eye color. The percentage of injected flies producing one or more rosy progeny (founders) is indicated along with the percentage of total progeny exhibiting rosy eyes. At least one progeny per founder was sequenced to determine if the targeted event had occurred. The percentage of founders in which the expected event occurred in one or more progeny is reported, as is the overall germline transmission rate (% injected flies yielding expected event). Note that while we did not recover the attP replacement of rosy, we did recover the precise deletion of the 6.1-kb rosy locus without attP incorporation in 1/18 crosses (7/1152 progeny) for an overall germline transmission rate of 5.6% for the deletion.

 

Table 2. chiRNA sequences used to target the rosy locus. Sequences of the five different chiRNAs used to disrupt rosy. Target strand orientation is relative to the gene. The site of the Cas9-generated DSB is indicated relative to the rosy ATG translational start site.

Methods: For all experiments, DNA plasmids encoding Cas9 downstream of the hsp70 promoter were injected into preblastoderm embryos at 500 ng/mL. chiRNAs were injected as DNA plasmids for in vivo transcription under control of the snRNA:U6:96Ab promoter at 500 ng/mL total concentration (250 ng/mL each for experiments employing two chiRNAs). The ssODN donor template was injected at 100 ng/mL. rosy mutations were identified by phenotype and confirmed by PCR and sequence analysis.

 

July 8, 2013

Our Cas9 and chiRNA plasmids are now available through Addgene.

 

May 30, 2013

New flyCRISPR Discussion Group

Please post questions or tips to the flyCRISPR Discussion Group.

We'll answer any questions we can and hope this will serve a forum for the community to share ideas and insights.