This second volume provides new and updated methods detailing advancements in CRISPR-Cas technical protocols. Chapters guide readers through protocols on prime editing, base editing, multiplex editing, editing in cell-free extract, in silico analysis of gRNA secondary structure and CRISPR-diagnosis.   Authoritative and cutting-edge,  CRISPR-Cas Methods, Volume 2 aims to serves as a laboratory manual providing scientists with a  holistic view of CRISPR-Cas methodologies and its practical application for the editing of crop plants, cell lines, nematode and microorganism. The chapter "CRISPR/Cas9-mediated gene editing in human induced pluripotent stem cells" is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.