CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.
Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.
The
Trichophyton rubrum
genome contains six proteins containing two or more lysin M (LysM) domains. We have characterized two of these proteins, LysM1 and LysM2, and demonstrated that these proteins have the capacity to bind two substrates, chitin and N-linked oligosaccharides associated with human skin glycoproteins. We have characterized the individual LysM domains in LysM1, and shown that the protein contains two functional LysM domains. Each of these domains can bind to chitin, to N-linked oligosaccharides in human skin glycoproteins, and to N-linked oligosaccharides on fungal glycoproteins. We hypothesize that LysM proteins could provide the pathogen with three important functions. First, the
T
.
rubrum
LysM proteins could shield host cell wall chitin from the human immune system. Second, the LysM proteins could shield the pathogen’s glycoproteins from host degradation and immune surveillance. Third, the LysM proteins could help facilitate pathogen adhesion to human skin.
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