Person:

Esvelt, Kevin Michael

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology. DOI: http://dx.doi.org/10.7554/eLife.03401.001

CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells.

Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.