The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

Abstract

The CRISPR‐Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid‐guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence‐specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high‐throughput sequencing, the discoveries and engineering of various innovative CRISPR‐Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid‐guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non‐existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.

Diverse CRISPR-Cas complexes require independent translation of small and large subunits from a single gene

“…CRISPR-Cas systems are divided into different types depending on their cas genes and their mode of CRISPR interference, which in turn has given rise to an expanding and broad range of novel genetic engineering tools ( 7, 38, 39 ). The type I-D CRISPR-Cas system appears to be a hybrid of type I and type III systems, yet the type I-D Cascade, involved in binding target nucleic acids, remained uncharacterized.…”

CRISPR-based detection of SARS-CoV-2: A review from sample to result

“…Cas12 has emerged as an alternative for Cas9 due to its distinct features, such as the ability to target T-rich motifs and no need for trans-activating crRNA. To this date, various subtypes of Cas12 proteins have been discovered ( Wang et al, 2020a ). Table 2 summarizes various subtypes of Cas12 proteins and their characteristics.…”

CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions

“…The type V proteins members possess the RuvC nuclease domain instead HNH domain to identify the site-specific T-rich PAM (5′) sequence just upstream to target region. In addition, Cas12 nucleases built staggered DSBs distal to PAM sequence unlike to Cas9 that helped to generate the unique knock-in strategies for genome editing through HNEJ mechanism [ 78 ].…”

“…These two proteins also carried some unique features, unlike Cas9 like these proteins cleaved the ssRNA instead of DNA because they have no catalytical DNA domain. Recently, studies stated that Cas13 proteins possess two identified highly conserved prokaryotic and eukaryotic nucleotide-binding domains containing specific RNA cleavage site [ 78 , 83 ]. Zhao et al [ 84 ] stated that bacterial Cas13a and CRISPR-RNA (crRNA) showed the successful knockout effects in mutant KRAS mRNA expression with the knockout efficiency of about 94%.…”