[Msi-workgroups-feedback] March Newsletter and Upcoming Events!

[Cyagen B. <Cya...@ma...>]

Cyagen Biosciences March 2016 Newsletter

Knock it down or knock it out?

In solving the mystery of gene function, there is no more important
clue than the phenotype of inactivating the gene of interest. With a
plethora of methods available, researchers must first determine what
approach is best for their specific scientific questions and
experimental systems.

For over a decade, RNA-interference-based methods of gene knockdown
(i.e. RNAi & shRNA) have provided a wealth of insight into gene
function, but in recent years the advent of CRISPR- and TALEN-based
methods now allow genome editing to be used to quickly and
efficiently test the effect of gene knockouts. Here, we review the
advantages and disadvantages of these approaches, and describe some
experimental situations in which one approach is better than another,
focusing primarily on CRISPR/Cas9 and shRNA. For a discussion of the
relative advantages of CRISPR/Cas9 versus TALEN, see our prior
Newsletter on this topic 

(http://cts.vresp.com/c/?CyagenBiosciences/8eaf8ca70a/e54c1e7a43/d6883c174d/utm_content=&utm_source=VerticalResponse&utm_medium=Email&utm_term=Text%20Version%20-%20Link%201&utm_campaign=March%20Newsletter%20and%20Upcoming%20Events%21.

-- shRNA Knockdown --
In this method, hairpin-forming RNAs are expressed from vectors
introduced into cells. These RNAs are cleaved into short (20-25nt)
double-stranded RNA molecules, which are then processed and
incorporated into the RNA-induced silencing complex (RISC) which then
complexes with target mRNAs to mediate mRNA degradation or
translation inhibition. The end result is down-regulation of gene
expression, without any change to the gene itself. Invariably, some
fraction of functional mRNA remains in the cell, so gene function is
not completely eliminated.

-Advantages-

*High transduction efficiency vector systems such as lentiviral shRNA
      vectors can be used to treat populations of cells, and for many
studies, data can be acquired directly, without the need for cloning.

*Due to efficient transduction and high shRNA expression from many
shRNA vector systems, experiments with shRNA vectors are generally
scalable and highly consistent when repeated.

*Some vector systems, such as regular plasmid shRNA vectors used in
transient transfections or piggyBac-based shRNA vectors, can be
removed from cells, making the knockdown reversible.

-Disadvantages-

*Incomplete loss of gene function due to remaining functional mRNA.

*Some vector systems, such as regular plasmid transfection, have
transient effects, rather than mediating permanent knockdown.


-- CRISPR Knockout --
In this method, a guide RNA (gRNA) homologous to an 18-22nt target
sequences in the genome is used to direct the coexpressed Cas9
nuclease to the target site. Hybridization of the gRNA localizes
Cas9, which then cuts the target site in the genome, generating
double-strand breaks (DSBs). Cellular repair of DSBs by the
nonhomologous end-joining pathway (NHEJ) generates stochastic errors
at the cut site. This usually results in small deletions, or more
rarely insertions and base substitutions. When these mutations
disrupt a protein-coding region (e.g. a deletion causing a
frameshift), the result is a functional gene knockout.

-Advantages-

*Changes to the genome introduced by CRISPR/Cas9 are permanent and
stable.

*Some clones should have a complete loss of gene function due to
frameshifts or premature stop codons in the open reading frame of all
copies of the gene.

*Different gRNAs can target Cas9 to different sites on the gene,
potentially generating a variety of informative mutations, such as
truncations.

-Disadvantages-

*The mutations introduced are stochastic in the cell population, so
individual clones need to be isolated and evaluated (e.g. by PCR or
sequencing) to determine the nature of mutations in each clone.

*In many cases, cells will have no loss-of-function mutations or
heterozygous mutations. In some cases this could be an advantage,
providing useful data for interpreting the knockout phenotype.


Cyagen Biosciences recently introduced VectorBuilder, an
award-winning online platform for custom design and cloning of DNA
vectors for all of your experimental needs, including a full line of
shRNA and CRISPR vectors, plus many other vector systems such as
lentivirus,adenovirus, AAV, piggyBac, regular plasmid and more! 
Cyagen Biosciences also providescustom virus packaging and mouse/rat
model generation, including transgenics, knockouts and knockins.


-- Upcoming Events --

Stop by and visit Cyagen at these events coming soon to an
institution near you!


*March 23  University of Iowa
*March 23  UCSD Moores Cancer Research Center
*March 24 Rockefeller University
*March 30 Texas A&M
*March 31 Texas Medical Center
*April 5 University of Texas Moffett Molecular Biology
*April 6 University of Texas MD Anderson Cancer Center

______________________________________________________________________
Click to view this email in a browser
http://hosted.verticalresponse.com/1154709/8eaf8ca70a/546866461/e54c1e7a43/

If you no longer wish to receive these emails, please reply to this
message with "Unsubscribe" in the subject line or simply click on the
following link: 

http://cts.vresp.com/u?8eaf8ca70a/e54c1e7a43/mlpftw

______________________________________________________________________
This message was sent by Cyagen Biosciences using VerticalResponse


Cyagen Biosciences
2255 Martin Ave.
Suite E
Santa Clara, California 95050
US


Read the VerticalResponse marketing policy: 
http://www.verticalresponse.com/content/pm_policy.html