Modeling human cancer predisposition syndromes using CRISPR/Cas9 in human cell line models

Abstract

The advancement of CRISPR mediated gene engineering provides an opportunity to improve upon preclinical human cell line models of cancer predisposing syndromes. This review focuses on using CRISPR/Cas9 genome editing tools to model various human cancer predisposition syndromes. We examine the genetic mutations associated with neurofibromatosis type 1, Li-Fraumeni syndrome, Gorlin syndrome, BRCA mutant breast and ovarian cancers, and APC mutant cancers. Furthermore, we discuss the possibilities of using next-generation CRISPR-derived precision gene editing tools to introduce a variety of genetic lesions into human cell lines. The goal is to improve the quality of preclinical models surrounding these cancer predisposition syndromes through dissecting the effects of these mutations on the development of cancer and to provide new insights into the underlying mechanisms of these cancer predisposition syndromes. These studies demonstrate the continued utility and improvement of CRISPR/Cas9-induced human cell line models in studying the genetic basis of cancer.

Keywords: CRISPR; iPSC; models.

Figure 1:

Next-generation methods to insert large DNA cargo into precise locations. A) Homing Endonuclease-assisted Large-sequence Integrating CAST complex (HELIX)-mediated insertion through TnsB-nAnil released cargo DNA and sgRNA guided CAST complex insertion. B) Programmable addition via site-specific targeting elements (PASTE)-mediated insertion via Bxb1-integrase activity between target attB insertion sites and attP site containing donor DNA. C) STRAIGHT-IN integration requires initial creation of AAVS1 landing-pad iPSC lines and Bxb1-mediated integration of cargo DNA, followed by excision of landing pad elements.

Figure 2:

Novel iterations of Prime Editor technology. A) Split-PE separates the MMLV reverse transcriptase from the canonical PE2 system. Deletions in the RNase H domain allow for packaging of Split-PE into two AAV vectors and subsequent delivery results in functional prime editing at low frequency. B) PEmax includes a codon optimized MMLV reverse transcriptase, additional mutations in SpCas9 to enhance activity, and a 3’ engineered pegRNA to protect from endonuclease activity. The transient expression of a dominant negative MLH1 protein acts to suppress DNA mismatch repair pathways from antagonizing the desired prime edit insertion.