Current trends of clinical trials involving CRISPR/Cas systems

Abstract

The CRISPR/Cas9 system is a powerful genome editing tool that has made enormous impacts on next-generation molecular diagnostics and therapeutics, especially for genetic disorders that traditional therapies cannot cure. Currently, CRISPR-based gene editing is widely applied in basic, preclinical, and clinical studies. In this review, we attempt to identify trends in clinical studies involving CRISPR techniques to gain insights into the improvement and contribution of CRISPR/Cas technologies compared to traditional modified modalities. The review of clinical trials is focused on the applications of the CRISPR/Cas systems in the treatment of cancer, hematological, endocrine, and immune system diseases, as well as in diagnostics. The scientific basis underlined is analyzed. In addition, the challenges of CRISPR application in disease therapies and recent advances that expand and improve CRISPR applications in precision medicine are discussed.

Keywords: COVID-19; CRISPR; HIV; cancer; clinical trials; gene therapy; genome editing; hemoglobinopathies.

Figure 1

CRISPR/Cas9 mediated adaptive immunity in bacteria. A typical CRISPR locus consists of a leader sequence followed by an array of short identical repeats interspaced by short unique spacer sequences, as well as a set of CRISPR-associated (cas) genes (5). Preceding the cas genes is the tracrRNA, which encodes a non-coding RNA that is complementary to the repeats. During the acquisition stage, foreign DNA was cleaved into short DNA fragments (protospacers) and incorporated into the CRISPR array in chronological order of invasion as a spacer (6). Once integrated, the new spacer is transcribed with all other spacers into a pre-crRNA. The tracrRNA is transcribed separately and combines with the repeat sequence of the pre-crRNA to form a heterodimer. Then, the heterodimer RNA is cut by RNase III to form mature crRNA (7). When the same foreign DNA invades again, the mature crRNA-tracrRNA structure engages the Cas9 protein to form an RNP. RNP guides the Cas9 protein to recognize the PAM sequence (NGG for SpCas9) of the foreign DNA by matching the crRNA with the exogenous genes and performing site-specific double-strand cleavage at the three bases upstream, then the foreign DNA sequence is destroyed (6).

Figure 2

Screening for global CRISPR clinical trials. We assessed clinical trials registered at ClinicalTrials.gov, ICTRP, and ICMJE. ^$Fifty-two and two duplicate CRISPR clinical trials were found in ICTRP and ICMJE, respectively, and were removed. *There were 20 records using ZFN (clinicaltrials.gov: 17, ICTRP: 2, ICMJE: 1) and 8 records using TALEN (clinicaltrials.gov: 8, ICTRP: 0, ICMJE: 0). ^#Basic study records and those not using CRISPR as a primary technique were excluded after being assessed for eligibility. Excluded trial identifiers: NCT03342547, NCT03450369, NCT04122742, NCT04478409, NCT05443607, NCT03681951, CTRI/2018/09/015807, CTRI/2021/09/036609, and CTRI/2023/09/057289.

Figure 3

Classification of CRISPR-based clinical trials. (A) A total of 84 clinical trials are classified according to disease categories; (B) among them, 29 trials and 55 trials are related to genetic diseases (35%) and non-genetic diseases (65%), respectively. We further divide the selected clinical trials into therapeutic trials and non-therapeutic trials. In genetic diseases, 5 trials are non-therapeutic, and the remaining 24 trials are therapeutic. Among the non-genetic diseases, 15 trials are non-therapeutic, and 40 trials are therapeutic.

Figure 4

Using CRISPR-Cas12a or Cas13a for precision diagnostics. Samples are extracted for DNA or RNA and amplified using RPA or RT-RPA, respectively. For Cas13a detection, the RPA products are further transcribed to RNA by T7 RNA polymerase. Then the amplified nucleotides are combined with Cas12a or Cas13a, crRNA, and an inactivated fluorescent ssDNA or ssRNA reporter. Upon crRNA binding and cleavage of the specific targeting sequence, the collateral activity of Cas12a or Cas13a will enable the cleavage of the inactivated reporter, resulting in activation of the fluorophore as an indicator for the presence of the target sequence (111, 112). Cas12a is an endonuclease with bilobed architecture, containing a recognition (REC) and a nuclease (NUC) lobe, the latter one harboring the wedge (WED), PAM-interacting (PI), bridge helix (BH), RuvC, and Nuc domains (113, 114). Binding of a single crRNA with the target strand (TS) induces conformational change and exposure of the catalytic site in the RuvC domain that cleaves the non-target strand (NTS) at 18 bp and TS at 23 bp downstream of the 5’-TTTV-3′ (V = A/G/C) PAM sequence sequentially to generate sticky ends (114). Subsequently, the release of the PAM-distal cleaved DNA keeps Cas12a catalytically active and allows collateral ssDNA trans-cleavage (115). F, fluorescence; Q, quencher. Cas13a is a single crRNA-guided RNase mediating ssRNA cleavage, consisting of a REC lobe and a NUC lobe. The REC lobe contains an N-terminal domain (NTD) and a Helical-1 domain. The NUC lobe contains two higher eukaryotic and prokaryotic nuclease (HEPN) domains, a linker that connects these two domains, and a Helical-2 domain (116). When the target ssRNA and crRNA are combined to form double-stranded RNA, the conformational change of the HEPN domains activates Cas13a, which relies on the protospacer flanking sequence (PFS, 3’ U, A, or C) to efficiently cut the targeted ssRNA and then non-specifically cut any ssRNA nearby (29).