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.

Keywords: CRISPR; Cas system; DETECTR; Genome engineering; SHERLOCK.

Fig. 1

CRISPR/Cas adaptive immunity system. The three stages such as CRISPR adaptation (stage 1), CRISPR RNA biogenesis (stage 2), and CRISPR interference (stage 3) are schematically illustrated. During the adaption stage, the injection of genetic material (virus) into bacterial cells triggers the Cas1 and Cas2 adaption module proteins, which cleaves the invading sequences (spacers) and are then incorporated into the CRISPR array. During CRISPR/RNA biogenesis stage, the CRISPR array is transcribed into a precursor to crRNA molecules (pre-crRNA), which are then cleaved into mature crRNAs. These mature crRNAs form effector complexes with Cas proteins. When foreign genetic material sequences match a CRISPR spacer, the matching crRNA binds to the invading strand and cleaves the invading strand with the help of Cas nuclease (CRISPR interference stage)

Fig. 2

Schematic illustration of CRISPR/Cas9 mechanism. A The Cas9 protein complex contains six domains (Recognition lobe (REC I), REC II, Arginine-rich bridge helix, PAM Interacting, HNH, and RuvC). REC I is the major domain responsible for binding with the gRNA, the REC II function is not studied. The arginine-rich bridge helix initiates cleavage activity upon binding to targeted sequences. The interaction with PAM confers PAM specificity, which is responsible for binding with the target sequence. The HNH and RuvC are nuclease domains to chop the target sequence. The Cas9 protein remains inactive due to the absence of gRNA. B The programmed gRNA binds to the Cas9 and generates changes in the protein, which leads the inactive Cas9 protein into its active form. Once triggered, it searches the target sequence by binding with a sequence that matches the PAM sequence (5′-NGG-3′). Then Cas9 generates DSBs at 3 bp upstream of the PAM using its HNH and RuvC domains

Fig. 3

Applications of CRISPR/Cas9 system. CRISPR/Cas9 system has revolutionized genome engineering: its accuracy, rapidity, and affordability permit its use in a nearly limitless range of applications. Since its discovery, researchers have been using the CRISPR/Cas9 system to cure diseases, discover new treatments, and for precision medicine. It does not stop there; beyond treating human diseases, CRISPR/Cas9 is also being utilized for studying the model and non-model insects’ biology, somatic genome editing, manufacturing biofuels, and engineering better crops (rice, wheat, etc.), etc. These advances made possible by the invention of the CRISPR/Cas9 system will change the lives of people globally

Fig. 4

Schematic illustration of CRISPR/Cas12 mechanism. The Cas12 protein requires only the crRNAs to generate DSBs. Cas12 protein cleaves the target region beside a PAM sequence (CTA, TTN, TTTN) with the help of the RuvC and nuclease lobe (NUC) domains. Once Cas12 starts encountering, it initiates R-loop, which forms base-pair hybridization between the crRNA and the target DNA strand. During this step, Cas12 matches the < 17 bp of the target sequence and leads to an R-loop formation. Once R-loop is formed, the Cas12 protein uses its active RuvC domain and generates a staggering cut in the non-target strand with the help of the PAM sequence

Fig. 5

a and b Mechanism of SHERLOCK and DETECTR systems. (A) Targeted double-stranded DNA (dsDNA) or RNA is amplified with recombinase polymerase amplification (RPA) or reverse transcription (RT)-RPA. The RPA is coupled with T7 transcription to covert targeted RNA for detection by Cas13 system. This amplification step with the combination of reporter probe, enable specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) to detect the targeted sequence, (B) In DNA endonuclease-targeted CRISPR trans reporter (DETECTR), DNA is amplified with RPA. The Cas12 system pairs with the single-stranded DNA (ssDNA) of interest, and the DNase activity of Cas12 system is initiated. This amplification step, combined with the reporter probe, enables DETECR to detect the targeted sequence

Fig. 6

Schematic illustration of CRISPR/Cas13a mechanism. Cas13a protein is activated through a single crRNA. Cas13a protein comprises crRNA, NUC lobes, and two nucleotide-binding (HEPN) RNase domains for targeting RNA. The Cas13a cleaves ssRNA, upon recognizing the target sequence (22–28 nt) complementary to the crRNA spacer. The target sequence is flanked by a protospacer-flanking site (PFS) at the 3′-end and crRNA binds together and cleaves the target region of ssRNA without the tracrRNA

Fig. 7

Schematic illustration of CRISPR/Cas13b mechanism. Cas13b protein is associated with the mature crRNA. This ÇRISPR/Cas13b complex searches for the target ssRNA and induces precise conformational changes at the ssRNA target with the help of the Protospacer flanking site (PFS), which flanks RNA targeting at the 5′ end and PAM sequence (NAN/NNA) at the 3′ end, resulting in nonspecific RNA cleavage

Fig. 8

Schematic illustration of CRISPR/Cas14 mechanism. The Cas14 protein comprises both tracrRNA and crRNA to target ssDNA. Cas14 protein recognizes the ssDNA with the help of tracrRNA and crRNA, mediates seed sequence interaction with the target ssDNA, and cleaves the ssDNA, not dsDNA or ssRNA. The cleavage efficiency of the Cas14 protein is more specific than Cas9, Cas12, and Cas 13 proteins without the presence of the PAM region