Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

Abstract

CRISPR-Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats -CRISPR-associated (Cas)) RNA guided endonuclease has emerged as the most effective and widely used genome editing technology, which has become the most exciting and rapidly advancing research field. Efficient genome editing by the CRISPR-Cas9 system has been demonstrated in many species, and several laboratories have established CRISPR-Cas9 as a screening tool for systematic genetic analysis, similar to shRNA screening. At least three companies have been founded to leverage this technology for therapeutic uses. To facilitate the implementation of this technology, many software tools have been developed to identify guide RNAs that effectively target a desired genomic region. Here, I provide an overview of the technology, focusing on guide RNA design principles, available software tools and their strengths and weaknesses.

Genomes in Focus: Development and Applications of CRISPR‐Cas9 Imaging Technologies

“…[91,92] What do these data mean in the context of genomic imaging?Collectively,they suggest that careful consideration should be given to off-target and chromatin effects when designing sgRNAs for imaging at arget locus.I fb inding at near-cognate sequences is indeed long-lived, guides should be algorithmically chosen to minimize off-target homology. [93][94][95][96][97][98][99] With respect to chromatin, the epigenetic state of the target locus may dictate the minimum number of guides that is required to achieve sufficient signal. Whether Cas9 can be rationally engineered to circumvent some of these challenges is atopic of ongoing research.…”

Genome im Fokus: Entwicklung und Anwendungen von CRISPR‐Cas9‐Bildgebungstechnologien

“…Zusammengenommen legen sie nahe, dass bei der Entwicklung von sgRNAs für die Abbildung eines genomischen Ortes sorgfältige Überlegungen zu Off‐Target‐ und Chromatineffekten angestellt werden müssen. Wenn die Bindung an nahverwandte Sequenzen tatsächlich langlebig ist, sollten die Guide‐Sequenzen algorithmisch so ausgewählt werden, dass die Homologie zu Off‐Targets minimiert wird . In Bezug auf Chromatin kann der epigenetische Zustand des Zielortes die minimal erforderliche Anzahl von Guide‐Sequenzen, die nötig ist um ein ausreichendes Signal zu erreichen, beeinflussen.…”

Ethical Issues in Genome Editing using Crispr/Cas9 System

“…The CRISPR/Cas9 system has many potential applications due to its ability to cut the DNA of any genome at any desired location by introducing the cas9 protein and appropriate guide DNA into a cell [7,8]. The system has the ability for genome editing and gene regulation in many types of organisms facilitating the function elucidation of target genes in biology and diseases.…”