FFRDC MOONSHOT PORTFOLIO TASK ORDER - CRISPR gRNA Reagents for NGCMs

Leidos Biomedical Research, Inc., Frederick MD

Investigators

Abstract

Next Generation Cancer Models (NGCMs) Most 2-dimentional cancer cell lines currently used to study the etiology of cancer, e.g. mechanism, treatment response, etc. inadequately model its genetic, epigenetic, and phenotypic complexity. Many lack molecular characterization of the original tumor’s genome, epigenome and transcriptome (and the case-matched normal DNA) as well as the clinical presentation, treatment, and outcome data. In addition, cell lines from certain subtypes (defined by pathology or molecular features) or populations, as well as rare and pediatric cancers, are underrepresented or not available. Reasonably robust protocols to establish the NGCMs from a number of tumor subtypes have been developed over the past several years. Organoid, conditionally reprogrammed cells (CRC), modified versions of those methods, and unique media formulations can be used to establish models, which appear to address current gaps in cancer models. Some protocols even allow co-culture of tumor and stroma tissues. Organoid culture methods were first created using stem cells from the mouse small intestine. The models usually consist of two or more cell types and develop structures that resemble in vivo organs, but lack innervation, blood supply, and mesenchyme. These three-dimensional structures are grown from epithelial stem cells and are amenable to expansion in culture under appropriate conditions. Cultures are grown in exogenous extracellular matrix using media that contain components that drive cell proliferation and differentiation, such as Noggin, R-spondin, Wnt, and epidermal growth factor. The method has been adapted to propagate other cell and tumor subtypes for many months which appear to be representative of the original tumors. Research is ongoing to determine if the NGCMs are immortal, genomically stable etc. CRC methodology was pioneered using human keratinocytes and subsequently expanded to other epithelial cell types. Propagation of primary or tumor human cells requires Rho kinase inhibitor and irradiated mouse fibroblast feeder cells. The CRCs proliferate indefinitely as stem-like cells but appear to maintain the ability to differentiate. Although they are typically cultured as monolayers, CRCs can also develop into organ-like three-dimensional structures when grown in a supporting matrix. New media formulations have also been very successful in establishing models from tumor types historically difficult to propagate in culture. As opposed to standard culture media that contain on average 40-50 ingredients, these new media have over 80 components. For example, using complex medium to establish cell lines from a diverse array of ovarian tumors, results in cultures that retain most of the genomic and molecular features of the tissue from which they originated. The NCI Office of Cancer Genomics (OCG), Center for Cancer Genomics, together with international institutions, established a consortium, the Human Cancer Models Initiative (HCMI) whole goal is to make available to the scientific community large numbers of the in vitro NGCMs from many tumor subtypes and patient ethnic groups that are not encumbered with excessive intellectual property (IP) constraints. The conditions used for propagation are freely available. Genomic Pertubagens Genetic methods are successful in exploring the mechanisms of disease and identifying novel therapeutic targets. The clustered regularly interspersed short palindromic repeats (CRISPRs) gene editing, a bacterial defense system against virus infection, is the basis of the newest mammalian genome editing technology. It consists of a DNA binding protein, usually a nuclease, and RNA(s), termed guide RNA (gRNA), that allows the DNA cutting to occur at the desired locus, though not necessarily in an exact location. One version of the method allows researchers to permanently modify genes in living cells. Since the discovery of this biological modification mechanism, research identified this defense system many in bacterial species and the nuclease protein can be quite distinct. In addition, a number of laboratories created “dead” nucleases which bind the DNA, but do not cut it and thereby can be used for transcriptional activation and inhibition without changing the genome. Each bacterium which uses the “CRISPR” system has its own nuclease and other requirements for function. The most commonly used enzyme for mammalian gene editing is Streptococcus pyogenes’ Cas9 (SpCas9) whose open reading frame (ORF) is 1368 amino acids. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving ‘blunt ends’ that often undergo changes/mutations as they are rejoined. Cpf1/Cas12a cuts DNA differently from Cas9 and is about 300 amino acids smaller. The Cpf1 system provides flexibility in choosing target sites. Similarly to Cas9, the Cpf1 complex must first attach to the protospacer adjacent motif (PAM, 2-6 base pair DNA sequence) and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from Cas9. The Cpf1 complex cuts in the DNA helix strands are offset, leaving short overhangs at the ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately. Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur. The gRNAs are very important for the specificity of the Cas binding. They are made up of 2 components, the sequence to which they will bind within the genome and protospacer adjacent motif (PAM). These PAMs are caspase dependent and affect specificity. A nuclease can be modified to reduce the PAM specificity/requirement. In addition, the nuclease editing of a gene provides more flexibility for gRNA binding. In summary, the NGCMs be utilized in research that will shed light on cancer mechanisms, response to novel treatments and even serve as patient avatars. Therefore, the experimental protocols to manipulate the NGCMs, the technologies and analytical methods need to be optimized for these applications. The goal of this RFP is to generate validated human editing reagents for all transcribed genes, i.e. “whole genome”, through a single construct of nuclease and gRNA(s).