CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis

Abstract

CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.

Keywords: CRISPR/Cas; bioanalysis; bioimaging; nucleic acid analysis; protein analysis.

Figure 2

The CRISPR imaging system for genomic imaging. It relies on dCas9 or gRNA-bound fluorescent substances to visualize genomic loci in living cells. (A) dCas9 binds to a fluorescent protein (FP) [31]. (B) The supernova labeling (SunTag) system binds multiple fluorescent proteins [36]. (C) The SunTag system binds to light-induced nuclear output tags (LEXY) [37]. (D) RNA aptamers bind to molecular beacons MB [39]. (E) sgRNA carries two different molecular beacons (MBs). (F) dCas9 combined with DQ for imaging [40].

Figure 1

Fundamental components of four CRISPR/Cas systems. (A) CRISPR/Cas9. (B) CRISPR/Cas12a. (C) CRISPR/Cas14(Cas12f). (D) CRISPR/Cas13a. Purple arrowheads indicate cis-cleavage sites, and black arrows indicate trans-cleavag.

Figure 3

Using RCas9 to target mRNA in living cells. (A) Schematic diagram of RNA imaging system using CRISPR/dCas9 [43]. The cross symbol represents sequences that do not contain the PAM sequence. (B) Schematic diagram of the CRISPR-Sunspot imaging strategy, depicting the utilization of three target sites within a single mRNA for visualization purposes [45]. (C) Schematic representation of the Pepper530 complex [46].

Figure 4

Targeting mRNA in living cells. (A) Strategy for imaging NEAT 1, SatIII, MUC 4, and GCN 4 RNAs in live cells using the CRISPR/dCas13 system [47]. (B) Development of RNA imaging strategy by a combination of Tat peptide with fluorescent RNA aptamers (TRAP-tag) [49]. (C) Schematic diagram of applying CRISPR-TRAP-tag to track SatIII lncRNA under SA treatment [49]. (D) Imaging demonstrating the localization and expression patterns of each mammalian construct [48]. Scale bars, 10 μm. (E) Schematic of CRISPR/dCas13 system binding with sgRNA containing [66].

Figure 5

Nucleic acid detection based on CRISPR/Cas9 system. (A) Nucleic acid detection based on NASBACC [92]. (B) CAS-EXPAR nucleic acid analysis system [102]. (C) CRISPR/Cas9 nucleic acid analysis system in conjunction with E-DNA sensors [104].

Figure 6

Nucleic acid analysis system based on Cas12. (A) Schematic of direct nucleic detection acid using HOLMES system [90]. (B) Schematic of direct nucleic acid using DETECTR system [24]. (C) Schematic of direct nucleic acid using HOLMESv2 system [105]. (D) Double-amplification sensing strategy based on terminal deoxynucleotide transferase (TdT) and CRISPR/Cas12a [98]. Scissors represent nicking endonuclease (Cas12a) cleaving the reporters.

Figure 7

Schematic diagram of nucleic acid detection system based on Cas13. (A) Schematic of ZIKV RNA detection by SHERLOCK [30]. (B) Schematic of direct viral detection using HUDSON and SHERLOCK [91]. (C) Overview of Cas13-based CREST modifications [89]. (D) Schematic of complementary Cas13 and Cas12a enzymes in 4-channel in-sample multiplexing [93].

Figure 8

CRISPR protein analysis system with fluorescence sensor. (A) Schematic of the TdT-combined CRISPR/Cas12a method for UDG activity assay [108]. (B) Schematic of a Cas13a/crRNA-mediated, RNA-triggered signal amplification system following DNA transcription [134]. (C) Schematic of a DNA-regulated CRISPR/Cas12a biosensor designed to detect non-nucleic acid targets. This biosensor combines enhanced upconversion LRET with luminescence amplification facilitated by a biomimetic PC chip [139].