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ICYMI - Summaries of Selected Publications
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New tools for genome modification

Daniel T. Grimes

 



Cas9 is a dream-come-true for genetic engineers: a targetable nuclease that will cause mutations and promote the knock-in of template DNA. But Cas9 has more tricks than that. It is possible to tag Cas9 at the N- or C-terminus with other proteins and, unperturbed by this additional weight, Cas9 will carry those protein activities with it to engineer-specified locations in the genome. Two recent papers have taken advantage of this to further expand our abilities to modify the zebrafish genome.

 
ExoCas9 generates larger deletions than Cas9.                           Figure from Clements et al., 2017 Genesis

In the first, Daniel Wagner and colleagues at Rice University tagged Cas9 with an Exonuclease from E. coli. While Cas9 alone typically led to DNA deletions of 1-5 bps, the new Cas9-Exo tended to induce larger deletions, often more than 10 bp. The authors show that these larger deletions gave stronger loss-of-function phenotypes. This technique - named RICE CRISPR! - may also be applicable to the mapping of gene regulatory elements by deletion analysis.

Outline of the procedure reported in Zhang et al., 2017 Nature Communications

The second report, from the lab of Shuo Lin and colleagues at Peking University, described the use of a modified Cas9 which cannot induce double strand breaks, but which carries an activity that catalyzes site-specific base conversion (e.g. C-to-T or C-to-A) just upstream of an NGG or NGA PAM site. The authors showed that this technique, called base editing, can be relatively efficient. This tool can be used for engineering point mutations into the zebrafish genome, perhaps most useful for introducing patient-specific variants in disease modeling studies.



RICE CRISPR: Rapidly increased cut ends by an exonuclease Cas9 fusion in zebrafish.

Clements TP, Tandon B, Lintel HA, McCarty JH, Wagner DS.

Genesis. 2017 Jun 27. doi: 10.1002/dvg.23044.

PMID:28653435

Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Zhang Y, Qin W, Lu X, Xu J, Huang H, Bai H, Li S, Lin S.

Nat Commun. 2017 Jul 25;8(1):118. doi: 10.1038/s41467-017-00175-6.

PMID:28740134





 

Developing to see the light

José Pelliccia

 


New neurons added during human development associate with existing mature neuronal circuits in the brain to maintain some plasticity in an ever-changing world of new experiences. The zebrafish brain also continuously integrates these newborn neurons into existing neuronal circuits during development and growth. This conservation has allowed Boulanger-Weill et al. the ability to further study the morphological and functional development of immature neurons in the optic tectum using zebrafish. They demonstrated that dendritic arbors in newborn neurons became more complex throughout maturation, and displayed a GABAergic or glutamatergic identity at the end of maturation.Furthermore, early newborn neurons had a collective response to a visual stimulus, similar to functionally mature neurons, followed by functional maturity of the visual receptive field size a day later. However, the newborn neurons’ selectivity to direction and orientation of a visual stimulus took much longer to develop, extending beyond the stages they examined. The authors also showed that after becoming responsive to visual stimuli, newborn neurons established long-lasting connections with functionally mature neurons, which required retinal inputs for proper integration of newborn neurons into mature circuits. This work sets the stage for exploring further the mechanisms that underlie newborn neuron integration into mature circuits. 

 Image provided by Germán Sumbre



Functional Interactions between Newborn and Mature Neurons Leading to Integration into Established Neuronal Circuits.

Boulanger-Weill J, Candat V, Jouary A, Romano SA, Pérez-Schuster V, Sumbre G.

Curr Biol. 2017 Jun 19;27(12):1707-1720.e5. doi: 10.1016/j.cub.2017.05.029.

PMID:28578928



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