Author: fillip

Temperature dependent activity of Cpf1

A few weeks ago Antonio Giraldez’s lab posted a new preprint on BioRxiv. The manuscript describes work by Miguel Moreno-Mateos and colleagues, including collaborators from Jennifer Doudna’s group, on the implementation of Cpf1 genome engineering in zebrafish and Xenopus. Cpf1 is an alternative RNA-guided endonuclease to Cas9, which has a number of distinct features that make it an interesting tool…

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pCFD4 PCR protocol

Xu Zhang from Frank Schnorrer’s lab in Munich has send me a kind email sharing his PCR protocol for pCFD4 cloning. This PCR, which introduces both guide sequences into this double gRNA vector, is prone to give background bands. I had several users write to me saying that they, sometimes exclusively, get a smaller band than the desired 600 bp…

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One day CRISPR workshop

The Congento initiative at the Champalimaud Center for the Unknown organises a one day CRISPR workshop in December. The focus is on applications to genetically tractable organisms and Frank Schnorrer from the MPI in Munich and I will talk about strategies to engineer the Drosophila genome. Registration is open until November 27th (here). Hope to see lots of you there!

Introducing the blog

If you visited crisprflydesign before, you might have realized that we have made very few changes to the website over the past 6 months. In particular the results and news section has been neglected. These sites were initially thought for the rapid sharing of results from ongoing experiments. I think that was very useful at the beginning, when the main…

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Differential activity of U6 gRNA plasmids

Fillip Port1, Hui-Min Chen2, Tzumin Lee2 and Simon Bullock1 1: MRC-Laboratory of Molecular Biology, Cambridge, UK 2: Janelia Farm Research Campus, HHMI, Ashburn, USA   Delivery of gRNAs for fly CRISPR/Cas genome engineering is most commonly done by either DNA or RNA injection or by creating transgenic gRNA lines.  Plasmids for both DNA injection and gRNA transgenes use polymerase III…

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Highly efficient targeting of yellow with transgenic act-cas9 and pU6-3gRNA

Hui-Min Chen and Tzumin Lee Janelia Farm Research Campus, HHMI Here we report a preliminary test experiment combining the act-cas9 (stock CFD-1) with two different gRNAs targeting yellow expressed from either the U6-2 or U6-3 promoter. The pU6-gRNAs were stably integrated into the genome and transgenic flies were crossed to CFD-1 flies. Offspring inherit a wildtype yellow allele from the…

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First test of stock CFD-5: UAS-cas9

Hui-Min Chen and Tzumin Lee Janelia Farm Research Campus, HHMI   We expressed UAS-cas9 (stock CDF-5) under the control of a germline Gal4 line and crossed these flies to transgenic flies expressing gRNAs targeting yellow under the control of the U6-3 promoter (pU6-3-gRNA y). The gRNA target site was: GGAGCGATATAGTTGGAGCCAGCTGG. Female offspring of this cross was then crossed to yellow…

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Inducing mutations in ebony with the act-cas9 flies

Fillip Port and Simon Bullock MRC Laboratory of Molecular Biology, Cambridge, UK The act-cas9 and nos-cas9 transgenics were generated in a yellow1 mutant background, making targeting yellow a little complicated and not ideal as a positive control. We therefore wanted to target another visible marker and settled for ebony, as it is very easy to identify during screening. This experiment…

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Targeting yellow with the act-cas9 flies

Fillip Port, Madalena Reimão-Pinto and Simon Bullock MRC-Laboratory of Molecular Biology, Cambridge, UK While making the cas9 transgenic lines we were very keen to test the system, so we started our first experiment with the very first transformants. Our injection of the act5-cas9 construct was quite successful and we got a good number of transgenic flies, which were used to…

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