🚀 go-pugleaf

Dear Colleagues,

As many of you know, we have previously made two solicitations to the
community regarding the construction of engineered zinc finger pairs
for endogenous zebrafish gene targets.  As a result of these efforts,
out of 96 zebrafish genes we have targeted to date, we have
successfully constructed zinc finger pairs for 49 zebrafish genes and
TALEN pairs for eight zebrafish genes.  Plasmids encoding these zinc
fingers and TALENs have been deposited with the non-profit plasmid
distribution service Addgene (http://www.addgene.org/zfc/arrays/ for
the zinc fingers and http://www.addgene.org/talengineering/TALENzebrafish/
for the TALENs).

We are writing again to solicit recommendations for appropriate genes
to target in this effort.  As we noted in a previous note to the
community, we plan to construct TALENs rather than zinc finger arrays
for this effort going forward.  We and others have recently shown that
transcription activator-like effector nucleases (TALENs) provide a
robust alternative for performing targeted gene knockout in zebrafish
(Sander & Cade et al., Nat Biotechnology 2011; Huang et al., Nat
Biotechnology 2011) and additional experiments from our groups and
others have also demonstrated that this platform has a high rate of
success and generates indel mutations in zebrafish with remarkably
high efficiency (Cade & Reyon et al., Nucleic Acids Res., in press;
Moore et al., PLoS One 2012).  The Joung lab has recently shown that
the simplicity of TALEN design allows these nucleases to be
constructed in high-throughput using an automated system and that the
targeting range of TALENs is essentially limitless (Reyon & Tsai et
al., Nat Biotechnology, 2012).

We plan to construct 96 TALEN pairs for this third solicitation.  Each
laboratory may submit up to TWO gene targets by completing an Excel
form that can be downloaded from here:  http://www.TALengineering.org/download.htm.
One gene recommendation should be placed on each of the "Sheets" in
the file.  Place your first choice gene on the first sheet and your
second choice gene on the second sheet.  Gene recommendations will
only be accepted via e-mail beginning at noon Eastern U.S. Time on
Friday, June 15th, 2012 (requests received before this date/time will
not be considered � please do not send requests before this).
Recommendations will be considered in the order in which they are
received and checked for appropriateness using the criteria described
below.  We will choose one gene for targeting from each lab until we
reach 96 gene targets.  If we receive fewer than 96 targetable "first
choice" gene requests, we will then consider �second choice� requests
in the order in which we received the e-mails until we reach 96 gene
targets.  Please note that we plan to make another solicitation later
in 2012.

For a gene to be considered appropriate for this effort, we ask each
recommender to verify on the form that mutations in the gene of
interest do not already exist and that efforts to generate mutations
in the recommended gene are not underway elsewhere.

If you have a gene that you would like to recommend for targeting with
TALENs, please complete the Excel spreadsheet form.  Please note that
ALL required fields must be filled out � we will not accept or
consider any incomplete forms.  Please e-mail the form to
zebrafishzfs@zincfingers.org at or after (and not before) 12:00 Noon
(Eastern US time) June 15, 2012.  Note that recommendations from
scientists at commercial entities will not be accepted.

VERY IMPORTANT NOTE:  Each gene submitted MUST include its Ensembl
Gene ID (ENSDARG) the Ensembl Transcript ID (ENSDART), and the gene
name (e.g.-- Example:  ENSDARG00000024771 , ENSDART00000033574 ,
slc24a5).  In the event that Ensembl has incorrectly annotated the
gene of interest, the full genomic sequence of your gene should be
included with exons denoted by upper case and introns in lower case.
Genes not correctly submitted in one of these formats will NOT be
considered for targeting.

With best regards,

Keith Joung
Randall Peterson
Joanna Yeh
Jeffry Sander
Deepak Reyon
Jon Foley
Massachusetts General Hospital