CRISPR therapeutic tools for complex genetic disorders and cancer (Review)

Abstract

One of the fundamental discoveries in the field of biology is the ability to modulate the genome and to monitor the functional outputs derived from genomic alterations. In order to unravel new therapeutic options, scientists had initially focused on inducing genetic alterations in primary cells, in established cancer cell lines and mouse models using either RNA interference or cDNA overexpression or various programmable nucleases [zinc finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN)]. Even though a huge volume of data was produced, its use was neither cheap nor accurate. Therefore, the clustered regularly interspaced short palindromic repeats (CRISPR) system was evidenced to be the next step in genome engineering tools. CRISPR-associated protein 9 (Cas9)-mediated genetic perturbation is simple, precise and highly efficient, empowering researchers to apply this method to immortalized cancerous cell lines, primary cells derived from mouse and human origins, xenografts, induced pluripotent stem cells, organoid cultures, as well as the generation of genetically engineered animal models. In this review, we assess the development of the CRISPR system and its therapeutic applications to a wide range of complex diseases (particularly distinct tumors), aiming at personalized therapy. Special emphasis is given to organoids and CRISPR screens in the design of innovative therapeutic HIV, human immunodeficiency virus; ICF, immunodeficiency centromeric region instability facial anomalies syndrome; iPSCs, induced pluripotent stem cells; IDLV, integrase-defective lentiviral vector; LAM-PCR, linear amplification-mediated PCR; LNPs, lipid nanoparticles; LTR, long terminal repeats; HTGTS, highthroughput genome-wide translocation sequencing; HD, Huntington's disease; HBB, hemoglobin subunit beta; HTT, Huntingtin gene; GUIDE-seq, genome-wide unbiased identification of DSBs enabled by sequencing; LCA10, Leber congenital amaurosis 10; MAPK, mitogen-activated protein kinase; Mll3, mixed lineage leukemia 3; MGEs, mobile genetic elements; MOI, multiplicity of infection; NHEJ, non-homologous end joining; NSCLC, non-small cell lung cancer; PI3K, phosphoinositide 3-kinase; Pten, phosphatase and tensin homolog; PanINs, pre-invasive pancreatic intraepithelial neoplasms; PAM, protospacer adjacent motif; RFLP, restriction fragment length polymorphism; RNPs, ribonucleoproteins; RFN, RNA-guided FokI-dCas9 nuclease; RNA-seq, RNA sequencing; RCT, rolling circle transcription; SCD, sickle cell disease; sgRNA, single-guide RNA; SNPs, single nucleotide polymorphisms; ssRNA, single-strand RNA;SSCs, spermatogonial stem cells; TCRs, T cell receptors; TRAC, T cell receptor alpha constant; 3D, three-dimensional; tracrRNA, trans-activating RNA;TALEN, transcription activator-like effector nuclease; TGF, transforming growth factor; TSS, transcriptional start site; TKIs, tyrosine kinase inhibitors; UGI, unique guide index; vQS, viral quasispecies; ZNFs, Zinc finger nucleases.