A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection

“…Secondly, Cas9 nuclease is applicable to a broad temperature range, whereas Cas12b nuclease has a higher temperature requirement; therefore, the optimal dissociation temperature for Cas12b nuclease is greater than 40 °C. When the reaction temperature was 37 °C, wild-type BhCas12b preferentially cut complementary DNA without forming double-strand breaks, resulting in a decrease in editing efficiency, which was hypothesized to be due to the difficulty of target DNA accessing the RuvC site of BhCas12b [ 56 ], as well as the difficulty of target DNA accessing the RuvC site of BhCas12b. By mutating BhCas12b, researchers increased the DNA’s affinity for the Cas mutant protein, thereby removing the reaction temperature restriction on the use of Cas12b in mammals [ 28 ].…”

Recent Progress in Prompt Molecular Detection of Exosomes Using CRISPR/Cas and Microfluidic‐Assisted Approaches Toward Smart Cancer Diagnosis and Analysis

“…Since this test relates to the identification of the human genome, it is likely to be used to diagnose cancer at various stages, particularly in the initial steps of incubation. [161,187,188] Lu et al investigated a CRISPR-Cas12a-based fluorescence bioassay by coupling advanced technologies, including terminal deoxynucleotidyl transferase (TdT), magnetic nanoparticles (MNP), and strand displacement (SDR) to detect lung cancer-induced exosomal miR-21. They found that the CRISPR-Cas12a-based fluorescence bioassay against mismatched sequences had a high specificity as well as a high sensitivity of about 161 fM for the determination of miR-21.…”

“…These tests are also completed swiftly. Since this test relates to the identification of the human genome, it is likely to be used to diagnose cancer at various stages, particularly in the initial steps of incubation [161,187,188] . Lu et al.…”

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

“…22 Third, both Cas12 and Cas13 are widely exploited in POC tests because of their unique trans-cleave ability, but they have different cleavage preferences: Cas12 can recognize and hydrolyze target DNA, and the recognition usually requires a PAM sequence; on the contrary, Cas13 does not need a PAM sequence, but can only recognize and hydrolyze ssRNA. 23 Thus, Cas12 and Cas13 can be utilized to detect different kinds of targets. In addition, due to their different cleavage objects, Cas12 and Cas13 systems can also be combined within one system to orthogonally test dual targets.…”