Design, Construction, and Validation of Targeted Gene Activation with TREE System in Human Cells.

Genome editing technologies can be diverted into artificial transcription activators. In particular, researchers have improved dCas9-based technologies by tandem-fusing or trans-accumulating effector domains. Previously, we developed a hierarchical effector accumulation system named "TREE," enabling robust activation of target genes even when strongly silenced. In this chapter, we describe our protocol to design, construct, and validate the TREE-mediated target gene activation in cultured human cells.

Various strategies of effector accumulation to improve the efficiency of genome editing and derivative methodologies.

CRISPR-Cas9 is a sophisticated tool in which Cas9/sgRNA complexes bind to the programmed target sequences and induce DNA double-strand breaks (DSBs) enabling highly efficient genome editing. Moreover, when nuclease-inactive Cas9 (dCas9) is employed, its specific DNA-binding activity provides a variety of derivative technologies such as transcriptional activation/repression, epigenome editing, and chromosome visualization. In these derivative technologies, particular effector molecules are fused with dCas9 or recruited to the target site. However, there had been room for improvement, because both genome editing and derivative technologies require not only the DNA-binding tools but also the additional components for their efficient and flexible outcomes. For genome editing, DSB repair molecules and knock-in donor templates need to act at the DSB sites. Derivative technologies also require their various effector domains to be gathered onto the target sites. Recently, many groups have developed and utilized inventive platforms to accumulate these additional components to the target sequence by modifying Cas9 protein and/or sgRNA. Here, we summarize the strategies of CRISPR-based effector accumulation and the improved methodologies using these creative platforms.

Biased genome editing using the local accumulation of DSB repair molecules system.

Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of microhomology-mediated end-joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, independently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor.

Three-Component Repurposed Technology for Enhanced Expression: Highly Accumulable Transcriptional Activators via Branched Tag Arrays.

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans, can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation system achieved by fusing GCN4 repeat to MS2 coat protein to accumulate numbers of activators, fused to scFv antibodies. By targeting the CDH1 gene, we show that our novel system, named "TREE," results in a greater effect of activating exogenous reporter and endogenous gene. Moreover, by targeting another gene, RANKL, we consistently show the superiority of the TREE system with fewer single-guide RNAs compared to conventional systems. Our TREE system is a promising tool for transcriptional activation and can potentially contribute to other dCas9-mediated technologies such as epigenome editing and chromosome visualization.