Easy and Efficient Human Genome engineering with CRISPR-Cas

Researcher can easily edit multiple spots at the same time in human genes. Other scientists believe that there are identified genetic changes that can be made to enable significant anti-aging treatments using genome editing.

1. Nature Scientific Reports – Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system

Here, researchers report an easy and efficient construction system for all-in-one CRISPR/Cas9 vectors expressing Cas9 protein and up to seven gRNAs. The expression cassettes of the gRNAs are tandemly ligated into a single vector using the Golden Gate cloning method. Furthermore, they demonstrate simultaneous multiple targeted mutagenesis and chromosomal deletions mediated by not only Cas9 nuclease, but also Cas9 nickase. In Cas9 nuclease-mediated genome editing, we constructed an all-in-one vector expressing Cas9 nuclease and seven gRNAs, and targeted seven genomic loci. In Cas9 nickase-mediated genome editing, they constructed an all-in-one vector expressing Cas9 nickase and six gRNAs, and targeted three genomic loci.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system. Our system provides an efficient targeting strategy for multiplex genome/epigenome editing, simultaneous activation/repression of multiple genes, and beyond.

Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering.

2. Nucleic Acids Research – Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

Abstract

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

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