A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe type II clustered regularly interspaced short palindromic repeat (CRISPR) systems and the associated Cas9 nucleases have evolved in archaea and bacteria for sequence-specific recognition of DNA targets via a single-stranded RNA intermediate (). In an engineered version of the CRISPR system, the Streptococcus pyogenes Cas9 nuclease is directed by guide RNAs (gRNAs) targeting 20 bp sequences adjacent to a 5′-NRG-3′ sequence motif, and the resultant cleavage has been used to edit the genome in several species (). A mutated nuclease-inactive Cas9 (dCas9) regulates gene expression by physically blocking transcription or through fusion to a transactivator (VP64, Ω subunit of RNA polymerase) or repressor domain (KRAB, SID) (). While transgene overexpression has been used to achieve cellular reprogramming (), reprogramming via direct activation of an endogenous gene has only been recently demonstrated through the use of transcription activator-like effectors (TALEs) (). However, difficulty in designing and codelivering multiple TALE expression constructs precludes simple screening and multiplexed gene activation that is straightforward with the dCas9-VP64 system. In this study, we used dCas9-based transactivators combined with an efficient lentivirus-based gene delivery system to induce cellular reprogramming.