Date Published: March 30, 2017
Publisher: Public Library of Science
Author(s): Aaron P. Mitchell, Donald C Sheppard.
The nuclease that creates double-strand breaks has two components: the Cas9 protein and a single guide RNA (sgRNA) . The commonly used CAS9 gene originated in Streptococcus pyogenes, and it has been modified for use in eukaryotic cells through inclusion of gene segments for a nuclear localization sequence and, in many cases, an epitope tag. CAS9 genes used in most fungi come from versions that were codon-optimized for human cells [6,7,8,9,11,12]; the CAS9 gene used in Ca. albicans was modified to accommodate the species’ variant genetic code . In all described human fungal pathogen Cas9 systems, the modified CAS9 gene is expressed constitutively from a fungal RNA polymerase II promoter [6,7,8,9,11,12].
In many systems, the Cas9 and sgRNA genes are carried on one or two cassettes that are integrated into a genomic site or, if the organism permits, present on plasmids. One generally useful approach is to first create a strain that expresses Cas9, then follow up with a second transformation that introduces an sgRNA gene or in vitro-synthesized sgRNA and, if desired, a repair template [6,7,9,11,12]. It has been demonstrated rigorously that Cas9 expression is innocuous in A. fumigatus, Ca. albicans, and C. neoformans [6,9,11]. A single Cas9-expressing strain can thus be used to create mutations in diverse genes that are determined by the choice of sgRNA and repair template.
Some of the most burning questions in the field of genome editing have to do with off-target effects [1,16]: How can off-target effects be assessed? How can they be minimized? Many off-target sites in other systems have one or a few mismatches to the sgRNA, particularly in its 5′ region, so mutations in candidate off-target sites may be tested through sequencing or nuclease-based assays . The capture of break sites for sequencing or whole-genome sequencing are also useful approaches . Technologies to minimize off-target effects are now maturing. For example, the use of paired Cas9 mutants that nick only a single strand can improve specificity considerably , and high-specificity Cas9 variants have been engineered recently . Alternatively, use of 5′-truncated sgRNAs has been shown to improve cleavage site specificity . It is encouraging that a 5′-truncated sgRNA has been shown to function for on-target effects in A. fumigatus . However, there have been few assessments of off-target effects in the human fungal pathogens. Interestingly, this field has a long-standing tradition of concern about secondary mutations and their phenotypic impact. Investigators have addressed this concern through analysis of complemented derivatives of mutant strains. Thus far, only one Cas9-sgRNA—directed mutation in a human fungal pathogen has been subjected to validation through complementation . To be fair, the test loci used for development of Cas9-sgRNA systems have been well characterized previously, and the newly created Cas9-sgRNA—directed mutations generally have the expected phenotypes. However, the development of facile complementation or gene-reconstitution systems to accompany genome editing technology will be critical to address newly accessible questions about gene functions and interactions.