Insight into the natural regulatory mechanisms and clinical applications of the CRISPR-Cas system

Abstract

CRISPR-Cas, the adaptive immune system exclusive to prokaryotes, confers resistance against foreign mobile genetic elements. The CRISPR-Cas system is now being exploited by scientists in a diverse range of genome editing applications. CRISPR-Cas systems can be categorized into six different types based on their composition and mechanism, and there are also natural regulatory biomolecules in bacteria and bacteriophages that can either enhance or inhibit the immune function of CRISPR-Cas. The CRISPR-Cas systems are currently being trialed as a new tool for gene therapy to treat various human diseases, including cancers and genetic diseases, offering significant therapeutic potential. This paper comprehensively summarizes various aspects of the CRISPR-Cas system, encompassing its diversity, regulatory mechanisms, its clinical applications and the obstacles encountered.

Keywords: CRISPR-Cas; Clinical applications; Genome editing; Regulatory mechanism.

Fig. 1

The CRISPR-Cas immune system overview involves several steps. In the CRISPR-Cas system, Cas1 and Cas2 proteins capture and integrate invading genetic material into the CRISPR array, which is then transcribed into crRNAs. These crRNAs, along with Cas effector proteins, target and degrade matching sequences in the interference stage.

Fig. 2

(A) Schematic of the editing strategy for TDT and SCD; the sgRNA-targeted editing sites at the BCL11A erythroid-specific enhancer region, with the five exons of BCL11A labeled in gold boxes, GATA1 indicating the GATA1 transcription factor binding site, and PAM denoting the NGG DNA sequence after the Cas9 target DNA sequence; (B) The editing strategy for LCA10 involves flanking the IVS26 mutations with CEP290 gRNAs 323 and 64, leading to productive editing through the deletion or inversion of the intervening sequence; (C) The schematic of ABE for CD3d SCID shows that correcting the disease-causing defects is possible by developing ABE.

Fig. 3

Ex vivo CRISPR engineering of human T cells for adoptive T cell therapy; Allogeneic and autologous T cells can be tested to investigate the effect of CRISPR technology in tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells.

Fig. 4

(A) The SHERLOCK assay steps consist of pre-amplifying the input DNA or RNA target, converting it to RNA through T7 transcription, and subsequently recognizing and binding by the Cas13-crRNA complex, which activates RNAse activity and results in the unquenching of the fluorescent RNA reporter; (B) the DETECTR assay involves extracting DNA, pre-amplifying it, and mixing it with CRISPR components, including Cas12 protein and a single-guide RNA (sgRNA), along with a fluorescent reporter. When the target DNA is introduced, Cas12 binds and activates, cleaving the reporter and releasing a fluorescent signal.