Cornerstones of CRISPR–Cas in drug discovery and therapy

Abstract

The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of personalized and reference genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders. Here we discuss how CRISPR-Cas can impact the next generation of drugs through accelerating the identification and validation of high-value targets, uncovering high confidence biomarkers and developing differentiated breakthrough therapies. We focus on the promises, pitfalls and hurdles of this revolutionary gene editing technology, and also discuss key aspects of different CRISPR-Cas screening platforms and offer our perspectives on the best practices in genome engineering.

Loss of Zbtb32 in NOD mice does not significantly alter T cell responses.

“…Directly targeting mutations into the NOD genetic background via CRISPR gene editing in NOD embryos is now possible 20 . Matching sgRNAs in exon 2 of Zbtb32 ( Figure 1A and B) and CAS9n were injected into 2 cell-stage embryos.…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…Only recently discovered, CRISPR/Cas9 technology has already been enhanced to the point of fulfilling most of the genome editing and gene regulation currently demanded, ranging from the ability to perform multiple gene insertions, gene knockouts, combinatorial libraries, to advanced fine-tuning of biosynthetic pathways [23,39,47,95]. However, off-targeting remains an important limitation to the Retrotransposons are composed of similar DNA sequences, which ultimately allows to generate a promiscuous gRNA able to target several of these targets at once.…”

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells

“…Moreover, the introduction of double-strand breaks by any method can be coupled with homology directed recombination (HDR) using DNA fragments or ssDNA oligonucleotides to introduce known allelic variations into specific sites in the genome. The consequence of these technological advances means that specific nucleotide changes can be engineered into any iPSC line to recapitulate the mutations causing any inborn error of hepatic metabolism no matter how rare 36 .…”

“…The efficiency of using CRISPR/Cas9 coupled with homology directed repair is likely to substantially increase as new approaches are described, and existing techniques are optimized 36 . With advances in next generation sequencing, new allelic variations of uncertain significance are being identified that correlate with liver disease.…”