CRISPR/Cas9 as a therapeutic tool for triple negative breast cancer: from bench to clinics

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) is a third-generation genome editing method that has revolutionized the world with its high throughput results. It has been used in the treatment of various biological diseases and infections. Various bacteria and other prokaryotes such as archaea also have CRISPR/Cas9 systems to guard themselves against bacteriophage. Reportedly, CRISPR/Cas9-based strategy may inhibit the growth and development of triple-negative breast cancer (TNBC) via targeting the potentially altered resistance genes, transcription, and epigenetic regulation. These therapeutic activities could help with the complex issues such as drug resistance which is observed even in TNBC. Currently, various methods have been utilized for the delivery of CRISPR/Cas9 into the targeted cell such as physical (microinjection, electroporation, and hydrodynamic mode), viral (adeno-associated virus and lentivirus), and non-viral (liposomes and lipid nano-particles). Although different models have been developed to investigate the molecular causes of TNBC, but the lack of sensitive and targeted delivery methods for in-vivo genome editing tools limits their clinical application. Therefore, based on the available evidences, this review comprehensively highlighted the advancement, challenges limitations, and prospects of CRISPR/Cas9 for the treatment of TNBC. We also underscored how integrating artificial intelligence and machine learning could improve CRISPR/Cas9 strategies in TNBC therapy.

Keywords: CRISPR/Cas9; drug resistance and artificial intelligence; gene editing; immunotherapy; triple negative breast cancer.

FIGURE 1

Overview of CRISPR/Cas9 (A). Components of the CRISPR/Cas9 system: (i). Cas9 endonuclease which is responsible for cleavage of target DNA sequence, (ii) single guide (sg) RNA formed by the fusion of crRNA and tra-crRNA chimera. (B). Cas9 includes multiple components such as Rec I, Rec II, NUC lobe (HNH and Ruv C are sub components) and a PAM interacting domain with their respective function, (C). CRISPR/Cas9 protein complex cleaves the DNA sequence into non-complementary and complementary form, and (D). CRISPR/Cas9 edit genome by following three steps: recognition, cleavage, and repair. The designed sg-RNA, guides Cas9 and recognizes desired sequence by crRNA, complementary base pair component. Cas9 recognized the PAM sequence at 5′-NGG-3′ and melt DNA by forming the DNA- RNA hybrid and activates for the cleavage. HNH domain of Cas9 cleaves the complementary strands and RuvC domain cleaves the non-complementary strands. CRISPR/Cas9 repair the dsDNA break by two pathways: Non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ repair dsDNA by an enzymatic process in the absence of exogenous homologous DNA, it is an error prone mechanism that can insert or delete the random DNA sequence. HDR is highly specific and requires homologous DNA templet.

FIGURE 2

CRISPR/Cas9 driven gene editing of various TNBC oncogenes leading to reduced tumor growth and metastasis. These oncogenes are CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 and CXCR7, Crypto1, ROR1, and ST8SIA involved in development and metastasis of TNBC.

FIGURE 3

CRISPR/Cas9 in immunotherapy to attack TNBC cells. The deletion of CDK5 and knockdown of PDL1 and CD155 enhance the immune system. Similarly, the deletion of A2AR and knockdown of TAA such as HER2, mucin 1 and TEM8 enhance the potency of CAR-T cells leaded to killing of cancer cells. CRISPR/Cas9 driven screening of T cells found disruption of p38 kinase, that enhance the anti-tumor activity of T cells. The modified T cells by CRISPR/Cas9 increased the expression pf TCR (T-cell receptors), leaded to anti-tumor activity of T cells.