Posters
« Back
Knockout of microRNAs using the CRISPR-Cas9 system with paired synthetic crRNAs
EP27373
Poster Title: Knockout of microRNAs using the CRISPR-Cas9 system with paired synthetic crRNAs
Submitted on 21 Mar 2018
Author(s): Eldon T. Chou, John A. Schiel, Elena Maksimova, Travis Hardcastle, Emily A. Anderson, Annaleen Vermeulen, Anja van Brabant Smith
Affiliations: Dharmacon part of Horizon Discovery Group
This poster was presented at Keystone Symposia - Noncoding RNAs: Form, Function, Physiology
Poster Views: 1,397
View poster »


Poster Information
Abstract: The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) system derived from Streptococcus pyogenes uses a Cas9 nuclease directed by a guide RNA (gRNA) to create a DNA double-strand break (DSB) at the target site. The gRNAs can be dual synthetic molecules, like the native bacterial system containing a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) (Figure 1), or a single synthetic guide RNA (sgRNA). The DSB is most often repaired by either nonhomologous end joining (NHEJ) or homology directed repair (HDR) through endogenous mechanisms within mammalian cells. NHEJ can result in insertions or deletions (indels) that produce functional gene knockouts through nonsense mutations or introduction of a stop codon. When using CRISPR-Cas9 components targeting coding genes, there are typically multiple protospacer adjacent motif (PAM) sequences (NGG for S. pyogenes) to choose from along the gene to design a gRNA. For most CRISPR Cas9 genome engineering experiments, one targeting gRNA is sufficient to generate the desired functional gene knockout. However, for some applications, it may be advantageous to use two gRNAs to generate a larger deletion and ensure gene knockout or to remove an exon, long non-coding RNA (lncRNA), or transcriptional regulatory element.Summary: We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.References: Report abuse »
Questions
Ask the author a question about this poster.
Ask a Question »

Creative Commons

Related Posters


Primary Human Hepatocytes 3D in vitro Culture Model for Studying Hepatic Function
Sujoy Lahiri, Julia Tritapoe, Kate Comstock, Michael F. Millett, Mark Kennedy, Theresa V. Nguyen, Deborah K. Tieberg, David T. Kuninger

FINGERPRINT 1H- NMR BASED ON THE CHEMICAL PROFILING OF COLOMBIAN RED PROPOLIS
Guillermo Salamanca Grosso; Monica Patricia Osorio Tangarife; David Garcia Mendez; Julien Wist

Delivery of RNP complexes for CRISPRi (dCas9) silencing in vitro and in vivo using crystalline ultrasmall particles (CUSP)
GM Unger*, VL Korman, BT Kren, DL Tobolt

Genetic Engineering in Male Sterility for Hybrid Variety Development
Abir Hasan Joy

VITVO: Mimicking In Vivo Complexity By The Innovative 3D Model
Olivia Candini1, Giulia Grisendi1, Elisabetta Manuela Foppiani1, Matteo Brogli1, Beatrice Aramini2, Valentina Masciale3, Carlotta Spano1, Tiziana Petrachi4, Elena Veronesi4, Pierfranco Conte5,6, Giorgio Mari1 & Massimo Dominici1,3