Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

Murine gammaherpesvirus 68 Rta-dependent activation of the gene 57 promoter.

The Rta homolog encoded by murine gammaherpesvirus 68 (gammaHV68) gene 50 is essential for virus replication and is capable of driving virus reactivation from the S11 latently infected B lymphoma cell line. Here we characterize Rta activation of gammaHV68 gene 57, which is abundantly transcribed during the early phase of virus replication. Infection of murine fibroblasts with an Rta null virus demonstrated that transcription of gene 57 is dependent on Rta expression. Analysis of the gene 57 promoter identified 2 distinct regions that are Rta responsive, either in the context of the gene 57 promoter or when cloned upstream of a heterologous promoter. Sequence analysis of these regions revealed homology to known Rta-responsive cis-elements in the closely related Kaposi's sarcoma-associated viral (KSHV) genome. In addition, two candidate binding sites for the cellular transcription factor RBP-Jkappa/CBF1 were also identified in one of the Rta-responsive regions, which may play a role in mediating Rta transactivation similar to that observed in some KSHV Rta-responsive genes. Overall, analysis of the gammaHV68 gene 57 promoter suggests that mechanisms of Rta activation are conserved among gamma2-herpesviruses.

Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis.

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the gammaHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type gammaHV68. From these analyses we identified 16 gammaHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other gammaHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.