Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy

Abstract

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) shows the opportunity to treat a diverse array of untreated various genetic and complicated disorders. Therapeutic genome editing processes that target disease-causing genes or mutant genes have been greatly accelerated in recent years as a consequence of improvements in sequence-specific nuclease technology. However, the therapeutic promise of genome editing has yet to be explored entirely, many challenges persist that increase the risk of further mutations. Here, we highlighted the main challenges facing CRISPR/Cas9-based treatments and proposed strategies to overcome these limitations, for further enhancing this revolutionary novel therapeutics to improve long-term treatment outcome human health.

Keywords: CRISPR; Cancer therapy; Cas9; Gene editing; Gene modification challenges.

Fig. 1

The stages of CRISPR/Cas adaptive immunity. The three phases of the CRISPR/Cas9 system are depicted schematically. When phage DNA is injected into a bacterial cell, the Cas1–Cas2 adaptation module proteins are activated, which remove spacer-sized segments of phage DNA and channel them into the CRISPR array. The CRISPR array is transcribed, and the resulting pre-crRNA is processed at repeat sequences to form crRNAs during CRISPR RNA biogenesis. The Cas protein effectors bind individual crRNAs. Effectors programmed by suitable crRNA attach to phage DNA with sequences matching a CRISPR spacer in the cell, and the resulting R-loop complex is destroyed by Cas executor nuclease

Fig. 2

Timeline highlighting main events of identification, CRISPR development (structural-functional relationships), applications, and CRISPR-based gene editing and clinical trials

Fig. 3

CRISPR/Cas9-mediated treatment has the potential to cure a variety of diseases. The number of diseases that CRISPR is now used to treat is rising by the day. The CRISPR/Cas9 system has been used to generate many disease-based models for many important human diseases, including viral diseases, neurological diseases, cancer, ocular disease, blood diseases, and cardiovascular diseases and disorders, as well as other complex genetic human diseases, according to data from clinical trials released recently

Fig. 4

Overview of CRISPR/Cas9-based gene editing of human iPSCs which includes both in vivo and in vitro methods. Gene editing techniques like CRISPR/Cas9 have allowed researchers to develop isogenic control human iPS cell lines to study the genetic pathways underlying disease and cellular function

Fig. 5

Challenges and overcoming strategies of CRISPR/Cas9. Immunogenicity, off-targeting, polymorphism, delivery technique, and ethical issues are main limitations, difficulties; and challenges of the CRISPR/Cas9 system in clinical trials and its recent discovery and usage in humans

Fig. 6

‘Immune-privileged’ sites and CRISPR/Cas9-mediating gene editing. Implementing the CRISPR Cas system for gene editing early in person’s life; and targeting immune-privileged organs are all attempts to overcome the limitations provided by immunogenicity against Cas9

Fig. 7

Nickase systems consisting of one or two nickases. H840 and D10 are two amino acids found in the Cas9 endonuclease protein that are involved in the cutting of one DNA strand by the enzyme. The RuvC domain contains the amino acid H840, while the HNH domain has the amino acid D10. The non-targeted strand is cleaved by Cas9 H840A, while the gRNA-targeting strand is cleaved by Cas9 D10A. Cas9 can only cut the strand complementary to the gRNA in a single nickase; however, a pair of sgRNA-Cas9n complexes can nick both strands at once (paired nickases). Additional concerns for gRNA design when using paired nickases include creating a 5’ overhang, the spacing between the two gRNAs, and the relative position of the two gRNA target sites

Fig. 8

Mechanisms of DNA repair outcomes of genome editing. Typically, DNA double-strand (ds) breaks caused by CRISPR/Cas9 are repaired via either homology-directed repair (HDR) or non-homologous end joining (NHEJ), depending on the circumstances. Exogenous ‘repair templates’ can be introduced into the genome by HDR, whereas NHEJ creates random insertions and deletions (indels) that can disrupt coding areas or catalyse genome rearrangements. The preference for HDR or NHEJ after DNA damage can be increased by small compounds that interfere with each system and so bias the cell toward one or the other after DNA damage

Fig. 9

DNA editing platform CHyMErA is a combinatorial system. Cell lines harbouring nuclear SpCas9 and LbCas12a, as well as a hgRNA expression cassette, provide the basis of the CHyMErA system. Cas12a gRNAs are fused with Cas9 and expressed under a single U6 promoter in hgRNAs. This process is completed by Cas12a, which identifies the direct repeat sequence and cuts upstream of it to release functional Cas9 and Cas12 gRNAs that can be loaded onto their respective nucleases for directed combinatorial genome editing