CRISPR/Cas systems usher in a new era of disease treatment and diagnosis

Fig. 1. Mechanisms of CRISPR/Cas system, BE and PE. A CRISPR/Cas system and NHEJ or HDR mechanisms of DSBs repair. The endonuclease Cas9 is directed to a specific genomic locus and cuts the double stranded DNA, targeted genes can be deleted and repaired via NHEJ. With a donor DNA template, targeted genes can be edited via HDR. B The mechanism of CBE. CBE mediates the direct conversion of cytidine to uridine even when located in different sequence motifs, resulting in a C·G to T·A transition. C The mechanism of ABE. ABE alters its base pairing preferences by yielding inosine. Inosine prefers to pair with guanosine under specific conditions, resulting in an A·T to G·C transition. D The mechanism of PE. PE is composed of a RT fused to Cas9 nickase and a pegRNA, mediates targeted indels and all 12 base-to-base conversions in mammalian cells

Fig. 2. Schematic diagram of viral vectors and non-viral vectors. Schematic of lentivirus and genetic components of lentiviral vectors. Two copies of positive-strand RNA are surrounded by the protein capsid and envelope; the therapeutic transgene is inserted between the two LTRs. Schematic of adenovirus and genetic components of adenoviral vectors. Double strand genome DNA is surrounded by the protein capsid and envelope; the adenoviral vector is stripped of all viral coding sequences, resulting in a vector with only 5’ and 3’ ITRs in addition to a packaging signal. Schematic of AAV and genetic components of rAAV vectors. A single-stranded genome is surrounded by the protein capsid; the therapeutic transgene, along with associated promoter and polyadenylation sequences, is inserted between the two ITRs. Schematic of lipid nanoparticles and genetic components of lipid nanoparticles vectors. The untranslated regions (UTRs) flank both the 5’ and 3’ ORFs, and genes encoding structural proteins are replaced by genes coding for proteins of therapeutic value. Schematic of eVLPs and genetic components of Hepatitis E virus (HEV)-like particles (HEV-LPs) vectors. Hepatitis E virus (HEV) is a liver…

Fig. 3. Ex vivo and in vivo strategies for therapeutic CRISPR genome editing. Ex vivo and in vivo strategies for therapeutic CRISPR genome editing. For ex vivo strategies, HSPs or T cells are extracted from patients, deliver the nuclease and therapeutic transgene to the cells by electroporation, viral vector or non-viral vector, then transduce cells with transgene and introduce modified cells back into patients

Fig. 4. Two strategies for CRISPR-based diagnostics and comparison and detection characteristics of the properties of CRISPR/Cas13/Cas12/Cas9 system. A Two strategies for CRISPR-based diagnostics. Left: Schematic of SHERLOCK and DETECTR. DNA or RNA is amplified by RPA or RT-RPA, respectively. T7-transcribed RNA is the amplified product by RPA, and the CRISPR/Cas13 (used in SHERLOCK) and CRISPR/Cas12 (used in DETECTR) systems bind of the crRNA to the complementary target sequence to activate the Cas enzyme and trigger collateral cleavage of quenched fluorescent reporters. Right: Schematic of NASBACC. RNA targets are amplified through NASBA, from reverse transcription (RT) to complementary DNA, a sequence-specific primer that appends a trigger sequence (purple) for the toehold sensor was used. B Comparison and detection characteristics of the properties of CRISPR/ Cas13/Cas12/Cas9 system. PAM: protospacer adjacent motif; PFS: protospacer flanking site; DSB: double-strand break