Cas3 Protein—A Review of a Multi-Tasking Machine

Abstract

Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation

“…Current evidence supports a model in which Cas3 nicks the DNA at the R-loop, loads onto the ssDNA, and processively unwinds and degrades DNA in a unidirectional and ATP-dependent manner ( Fig. 2A) (19,33,49). Structures of T. fusca Cascade bound to a DNA target and Cas3 revealed that a protruding "bubble" in the nontarget strand of the R-loop is required for Cas3 to nick the DNA ( Fig.…”

“…Formation of the R-loop induces a conformational change in the complex that enables recruitment of Cas3, a helicase-nuclease protein that is required for target DNA degradation ( Fig. 2A) (19,20,48). Current evidence supports a model in which Cas3 nicks the DNA at the R-loop, loads onto the ssDNA, and processively unwinds and degrades DNA in a unidirectional and ATP-dependent manner ( Fig.…”

“…The Type I Cascade has no intrinsic enzymatic activity, and relies on recruitment of Cas3, a helicasenuclease, to degrade dsDNA in trans (Fig. 1A) (19,20). Type III CRISPR-Cas Csm/Cmr possess intrinsic DNase and RNase activities, but also synthesize a second messenger, cOA, which binds and stimulates RNA cleavage by Csm6/Csx1, a separate nuclease effector ( Fig.…”

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

“…Another Cas protein is Cas3, and this Cas protein participates in the CRISPR intervention in the third stage of CRISPR immunity (He et al, 2020[ 45 ]) and is essential for phage defense in the CRISPR-Cas system (Jackson et al, 2014[ 57 ]). While scientific research on the CRISPR-Cas9 system continues, the importance of Cas proteins continues to be discovered.…”

A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity

“…Recognition of PAM and the ‘seed’ reaction by Cascade trigger formation of an R-loop (‘RNA-loop’) complex in which Cascade base-pairs crRNA with a ‘targeted’ strand of DNA duplex, and the other ‘non-targeted’ DNA strand is displaced as ssDNA [ 16 ]—the importance of R-loops in biology is reviewed recently in [ 17 ]. In CRISPR systems, Cas3 is recruited to the Cascade R-loop complex and degrades DNA, processes reviewed recently in [ 18 ]. Therefore, Cas3 destroys MGE DNA and in the process generates DNA fragments that may prime adaptation [ 19 ], by their capture and integration into CRISPR DNA, completing a virtuous cycle of CRISPR activities.…”