Development of CRISPR-Cas systems for genome editing and beyond

Abstract

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotechnology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstration of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

CRISPR-Cas12a-assisted PCR tagging of mammalian genes

“…These lesions can promote integration of the desired heterologous DNA sequences via DSB repair pathways such as homologous recombination (HR) or canonical non-homologous end joining (c-NHEJ) (Scully et al, 2019). While zinc finger nucleases and TALENs where initially shown to yield high on-target editing rates, the CRISPR-Cas endonucleases are nowadays preferred due to their simplistic usage and versatility (Zhang, 2019). Among different Cas-endonucleases, Cas9 has found its way into most genome engineering applications mainly because for historical reasons.…”

Inherited cardiac diseases, pluripotent stem cells, and genome editing combined—the past, present, and future

“…When the same hPSC-CM disease model was referred to in more than one publication, only the original reference that described either the isolation of the patient material or the generation of the line was included, unless a different type of control was used in the subsequent publication introduce or correct at a specific genetic location if such a motif is not within the same vicinity, although this has been addressed to some extent by the development of alternative Cas9 variants with varied PAM requirements. 75 Perhaps a greater impediment is inefficient on-target editing, with substantial variability even occurring between target sequences in proximity (ie, 10-20 nucleotides) to each other. These differences might be due to the local chromatin structure, with editing efficiencies reduced if the target sequence is bound by nucleosomes.…”

Evaluating the Probability of CRISPR-based Gene Drive Contaminating Another Species

“…So far, the CRISPR-Cas9 system has been shown to cut at target sites specified by the guide RNA in all species that have been tested, including animals, plants, bacteria and parasites (Zhang 2019) . We therefore consider here that the probability cut that the CRISPR-guide RNA recognizes and cuts at a DNA site in the new host is simply the probability that the host genome contains a site targeted by the CRISPR-guide RNA of the gene drive construct.…”

“…We therefore consider here that the probability cut that the CRISPR-guide RNA recognizes and cuts at a DNA site in the new host is simply the probability that the host genome contains a site targeted by the CRISPR-guide RNA of the gene drive construct. The target site recognized by CRISPR-Cas9 is 20 nucleotides long followed by a protospacer adjacent motif (PAM), typically NGG (Zhang 2019) . Importantly, studies in yeast and human cells indicate that CRISPR-Cas9 cleavage activity can still occur with three to five base pair mismatches in the 5' end (Fu et al 2013;Hsu et al 2013;Roggenkamp et al 2018) .…”

“…The Cas9 protein is derived from the bacteria Streptococcus pyogenes (Zhang 2019) and can trigger an immune response in mice (Chew et al 2016;Chew 2018) . During the gene drive multiplication process, germline cells produce Cas9 proteins to cut the DNA and insert the gene drive cassette (Fig.…”