Delivery Methods, Resources and Design Tools in CRISPR/Cas

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR associated system 9 (CRISPR/9) system was discovered as the bacterial immune system against invading viruses. Its applications not only include genome editing but it is also being used for gene regulation, epigenome editing, chromatin engineering, and imaging. The most widely used CRISPR/9 system which have been used for experimental genome editing consists of two components, i.e. 9 nuclease protein and sgRNA (a 20 nucleotide (nt) long RNA molecule). The programmability and specificity of this system is conferred by the gRNA molecule. These two components can either be delivered in the form of ribonucleoprotein complex (RNPs), in vitro transcribed mRNA or in the form of DNA which is transcribed in vivo to produce 9 and sgRNA. The efficiency of this system depends upon the proper delivery, efficient transcription, and optimal activity of sgRNA and 9 on intended target site and minimum unwanted genome perturbations, known as off-target effects. In this chapter, we will present a brief introduction and history of CRISPR/ followed by description of various tools used for its delivery and overview of gRNA design tools. Different resources are described which are used to validate the intended genome editing. Finally, we will conclude with future directions and the broader impact of CRISPR technologies.