Abstract
Establishment of production platforms through prokaryotic engineering in microbial organisms would be one of the most efficient means for chemicals, protein, and biofuels production. Despite the fact that CRISPR (clustered regularly interspaced short palindromic repeats)–based technologies have readily emerged as powerful and versatile tools for genetic manipulations, their applications are generally limited in prokaryotes, possibly owing to the large size and severe cytotoxicity of the heterogeneous Cas (CRISPR-associated) effector. Nevertheless, the rich natural occurrence of CRISPR-Cas systems in many bacteria and most archaea holds great potential for endogenous CRISPR-based prokaryotic engineering. The endogenous CRISPR-Cas systems, with type I systems that constitute the most abundant and diverse group, would be repurposed as genetic manipulation tools once they are identified and characterized as functional in their native hosts. This article reviews the major progress made in understanding the mechanisms of invading DNA immunity by type I CRISPR-Cas and summarizes the practical applications of endogenous type I CRISPR-based toolkits for prokaryotic engineering.
Highlights
Throughout the past billion years, bacteria and archaea have evolved a range of defense mechanisms to defend themselves against their viral predators (Doron et al, 2018), including restriction–modification systems, abortive infections and phage adsorption blocks, and the recently discovered CRISPR-Cas systems (Jansen et al, 2002)
All other tested 8-nt single-stranded DNA (ssDNA) oligos that matched the crRNA at a region outside the 1- to 8-nt stretch exhibited no measurable binding affinity. These results indicated that the 1- to 8-nt sequence within the crRNA, immediately adjacent to the protospacer adjacent motif (PAM), played an essential role in defining an invading DNA as an attacker
Because of the host-specific property of endogenous CRISPR-Cas systems, each of them must be thoroughly characterized in the host cells, including determination of PAM sequences, demonstration of Cas functions, and identification of mature crRNAs, as well as dissection of its mode of action during foreign DNA defense
Summary
Throughout the past billion years, bacteria and archaea have evolved a range of defense mechanisms to defend themselves against their viral predators (Doron et al, 2018), including restriction–modification systems, abortive infections and phage adsorption blocks, and the recently discovered CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPRassociated) systems (Jansen et al, 2002). Intensive investigations on model type I CRISPR-Cas systems have revealed molecular mechanisms of multi-Cas CRISPRbased antiviral defense, in respect of crRNA processing, effector complex assembly, PAM recognition and R-loop formation, and Cas3-executed DNA target destruction (Figures 1C,D). In type I-E Cascade of Thermobifida fusca, the Cas subunit played the same roles in specifying the PAMs, while through contacting the non-target strand (Xiao et al, 2017) In both cases, PAM sequences were recognized by the Cas proteins at the minor groove side, explaining the promiscuity of PAM recognition in these systems. Cascade binding could destabilize the target DNA duplex, allowing crRNA to first pair with the protospacer within the seed region and throughout the whole matching sequences and forming a full R-loop, where the non-target strand is bound by the Cas dimer (Hochstrasser et al, 2014; Szczelkun et al, 2014). This may indicate that unwinding DNA target by the ATP-dependent Cas helicase domain could further provide ssDNA substrates for the nuclease domain of Cas, or for other host nucleases, leading to degradation of the entire DNA target (Brouns et al, 2008; Mulepati and Bailey, 2013)
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