January 8th, 2021
Adenine Base Editors (ABE) convert adenine into inosine within ssDNA substrates, producing targeted A:T to G:C transition mutations. The catalytic subunit of ABE enzymes consist of an evolved tRNA adenosine deaminase (TadA) that was engineered to perform deamination of DNA substrates through the critical D108N mutation. Because natural TadA deaminates RNA molecules, it was later found that ABE retains high off-target activity against RNA molecules.
In this paper, Rallapalli et al. from the Kaushik Ganapathy, Alexis Komor and Francesco Paesani labs at UC San Diego use an elegant multidisciplinary approach including bioinformatics, biophysical techniques, MD simulations and experiments assays to study TadA/ABE activity on RNA. They use sequence-based entropy model to gain insights on the role played by each amino acid on the primary sequence of TadA.
Given that ABE catalyze A:T-to-G:C transitions, ABE enzymes have the potential to correct >50% of all pathogenic mutations identified in patients. This work deploys elegant strategies to gain insights into enzyme functions to engineer and improve enzymes complementary to classical mutagenesis-engineering approaches.
January 18th, 2021
Type-III CRISPR-Cas systems have the most complex mechanisms of action to provide immunity against invaders. Type-III CRISPR loci contain enzymes exhibiting both CARF (CRISPR-Cas Associated Rossmann Fold) domain, such as the important Csm6/Csx1 nuclease, and a domain with regulatory or catalytic function. Upon recognition of foreign invader's transcripts by Cas10/gRNAs effector complex, the targeted transcript is cleaved resulting in the activation of the ribonuclease Csm6. Csm6 is activated by 3'-5' cyclic oligoadenylate (cA) that bind its CARF domain, generating robust degradation of all foreign RNA transcripts. The presence of additional proteins with a CARF and catalytic domains in Type-III CRISPR loci raises questions on the possibility that other mechanisms of defense exist.
In this paper, Rostol, Xie et al., from the Luciano Marraffini lab investigate the function of a protein which contains both a CARF and an endonuclease-like domain, and which is associated with CRISPR loci. The authors show that this small protein (43.9KDa) degrades ssDNA and ssRNA upstream of T(A/G) sites in vitro upon activation by cA4, demonstrating that Card1 (cA-activated ssRNAse and ssDNAse 1) is a nuclease against ssRNA and ssDNA, but unlike Csm6 which only targets ssRNA. Next, the authors solved crystal structures of Card1 in different states, enabling atomic resolution of the protein. In particular, the crystal structures revealed that Card1 is a homodimer, the position where cA4 binds between the two domains and a possible mechanism of ssDNA/ssRNA cleavage. Thus, the authors tested the functional role of Card1 in immunity against plasmids and phages. They observed that the ssDNA activity (target plasmid was degraded) but not the ssRNA activity (transcriptome unchanged) is involved in immunity against plasmids in a Cas10-dependent manner. The authors propose that Card1 may introduce DNA lesions in the bacterial chromosome to induce cell dormancy. Finally, the authors confirm that Card1 has a role in anti-phage immunity in S.aureus through its RNA activity when it is programmed to target late phage transcripts, similar to Csm6.
This interesting work reveals the diversity of nucleases and associated activities involved in the war between phages and bacteria.
A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems published in Science
Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers published in Nature