Application of CRISPR for In Vivo Mouse Cancer Studies

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in cancer research to edit specific genes and study their functions. This applies both to in vitro and in vivo studies where CRISPR technology has accelerated the generation of specific loss- or gain-of-function mutations. This review focuses on CRISPR for generating in vivo models of cancer by editing somatic cells in specific organs. The delivery of CRISPR/Cas to designated tissues and specific cell compartments is discussed with a focus on different methods and their advantages. One advantage of CRISPR/Cas is the possibility to target multiple genes simultaneously in the same cell and therefore generate complex mutation profiles. This complexity challenges the interpretation of results and different methods to analyze the samples discussed herein. CRISPR-induced tumors are also different from classical tumors in pre-clinical models. Especially the clonal evolution of CRISPR-induced tumors adds new insight into cancer biology. Finally, the review discusses future perspectives for CRISPR technology in pre-clinical models with a focus on in vivo screening, CRISPR activation/inhibition, and the development of prime/ base-editing for the introduction of specific gene editing.

Keywords: CRISPR; CRISPR screening; CRISPRa; CRISPRi; base editing; gene delivery; indel; mouse models; pre-clinical models; prime editing.

Figure 1

Delivery of CRISPR/Cas to induce cancer in the mouse. Schematic overview of delivery of sgRNA and Cas protein to different mouse organs. Plasmid delivery has mainly been done to the liver but also to astrocytes, keratinocytes mussel and pancreatic cells by either intravenous injections or by electroporation. Lentivirus (LV) has been applied to deliver CRISPR to the liver, pancreas, breast, lung and brain cancer. Adeno virus (AV) has been used to induce lung, liver, and glioma cancers. Adeno associated virus (AAV) is used to induce lung, liver, glioma, and prostate cancers by delivery through injections, operation or inhaling. Lipo nano particles (LNP) have emerged for CRISPR/Cas delivery and have been applied to GBM, liver and lung cancer.

Figure 2

Two combination system for in vivo CRISPR editing. Combination of viral particles with transgenic mice for Cas9 expression has multiple advantages. Cas9 can be ubiquitously expressed [34] or controlled by Cre expression [10] or tetracycline [42]. Cas9 protein is not delivered by viral particles and does not take space from the viral genome. Furthermore, by separating Cas9 and sgRNA expression, biosafety is increased as self-inoculation will not contain a risk for CRISPR editing of the scientist.

Figure 3

Indel analysis of CRISPR induced tumors. (A) Base call from and edit samples and a control at the target site of the sgRNA (marked with a black line and PAM site with a red dash line). Preferable editing site is a 3+ of the sgRNA (black dash line). (B) Indel analysis of a tumor, which predicted to be clonal. The analysis shows that 34% of the genome is unedited. 32% of the target has 1 base pairs deletion and 30% has 2 base pairs deletion. This suggests that ~60% of the cells are edited at the target site and ~40% of the cells not edited and could be cells of the tumor microenvironment. (C) Indel analysis of a tumor, which predicted to be polyclonal. The analysis shows that 40% of the genome is unedited. Of the edit genome, different indel’s on 19% (−2 base parries), 12% (−5 base parries), 11% (+1 base pare) and 11% (−4 base parries). This suggests that the tumor contains two clones, or that genomic amplification has taken place.