Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing

Abstract

Artificial nuclease-dependent DNA cleavage systems (zinc-finger nuclease, ZFN; transcription activator like effectors, TALENs) and exogenous nucleic acid defense systems (CRISPR/Cas) have been used in the new era for genome modification. The most widely used toolbox for genome editing, modulation and detection contains Types II, V and VI of CRISPR/Cas Class 2 systems, categorized and characterized by Cas9, Cas12a and Cas13 respectively. In this review, we (1) elaborate on the definition, classification, structures of CRISPR/Cas Class 2 systems; (2) advance our understanding of new molecular mechanisms and recent progress in their applications, especially beyond genome-editing applications; (3) provide the insights on the specificity, efficiency and versatility of each tool; (4) elaborate the enhancement on specificity and efficiency of the CRISPR/Cas toolbox. The expanding and concerted usage of the CRISPR/Cas tools is making them more powerful in genome editing and other biotechnology applications.

Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length

“…In fact, we do not expect greatly improved specificity with respect to DNA binding and interference by Cas3, which, however, needs to be more thoroughly tested by assessing the effect of mismatches and other target alterations. An exception may be applications that require precise positioning of functional domains, such as transcriptional activators, dimerizing DNA nucleases, or base-editing enzymes (Guilinger et al., 2014, Tang and Fu, 2018, Shimatani et al., 2017). When fused to the PAM-distal end of Cascade, the required positioning of the domain with respect to the DNA should only be achieved for R-loops that extend over the full spacer.…”

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…The CRISPR systems are adaptive evolved for counteracting foreign DNA or RNAs, and the systems are present in nearly half of bacteria and almost all archaea (Grissa et al, 2007b;Zetsche et al, 2015a), but absent from eukaryotes or viruses (Jansen et al, 2002). The CRISPR/Cas systems have been categorized into two classes and six major types based on the constitution of effector protein and signature genes, protein sequence conservation, and organization of the respective genomic loci (Koonin et al, 2017;Tang and Fu, 2018). Among these CRISPR systems, the Cas9 (Type II), Cas12a (previously known as Cpf1, type V) and their mutant variants are most investigated effectors, and have shown broad applicational potentials in genome editing, gene regulation, DNA detection, DNA imaging, etc.…”

“…Among these CRISPR systems, the Cas9 (Type II), Cas12a (previously known as Cpf1, type V) and their mutant variants are most investigated effectors, and have shown broad applicational potentials in genome editing, gene regulation, DNA detection, DNA imaging, etc. (Tang and Fu, 2018;Miao et al, 2019).…”

“…The CRISPR system is prokaryotic adaptive immune system against intruded heterologous DNA/RNA from virus or other organisms (Grissa et al, 2007a;Sorek et al, 2013). So far, the CRISPR/Cas system has been intensively adopted as toolbox for both fundamental studies and biotechnological applications for genome editing, molecular diagnosis, metabolic engineering, gene function mining, etc., in microorganisms, plants and mammals (Sander and Joung, 2014;Zhang et al, 2014;Wang H. et al, 2016;Tang and Fu, 2018;Tarasava et al, 2018;Armario Najera et al, 2019;Moon et al, 2019;Xu and Oi, 2019). In the field of microbial biotechnology, the CRISPR/Cas system has been applied for numerous model and non-model microorganisms, e.g., Escherichia coli (Jiang et al, 2013), Saccharomyces cerevisiae (DiCarlo et al, 2013), Bacillus (Westbrook et al, 2016), Clostridium (Li et al, 2016;Joseph et al, 2018), Corynebacterium (Jiang et al, 2017), Lactobacillus (Oh and van Pijkeren, 2014), Mycobacterium (Choudhary et al, 2015), Pseudomonas (Tan S. Z. et al, 2018), Streptomyces (Cobb et al, 2015).…”

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

“…CRISPR‐Cas systems so far have been divided into Class 1 and Class 2 based on the constitution of effector proteins (Koonin, Makarova, & Zhang, 2017). Among them, those designated Class 2, characterized by only one effector protein, are the easiest to be implemented in gene editing as well as manipulation of cell‐free nucleic acids, thanks to their simplicity (Sashital, 2018; Tang & Fu, 2018).…”