331. Development of Neisseria meningitidis CRISPR/Cas9 Systems for Efficient and Specific Genome Editing

Abstract

Targeted genome modification using the CRISPR/Cas9 system has been extensively embraced by the research community as a powerful tool in biotechnology and life sciences, and it holds promise for the future of personalized medicine. The CRISPR/Cas9 system consists of an RNA guide strand (gRNA) for target site recognition and the CRISPR associated protein Cas9 for DNA cleavage. Cleavage of a site requires a protospacer adjacent motif (PAM) sequence. CRISPR induced DNA double-strand breaks (DSBs) stimulate cellular DNA repair via non-homologous end joining or homologous recombination, which allows specific genome modification. However, previous studies have demonstrated high levels of off-target cleavage with CRISPR/Cas9 that may lead to unwanted or deleterious genomic modifications. Therefore, developing strategies to reduce nuclease off-target effects is essential for therapeutic applications of genome editing.The widely used Streptococcus pyogenes (Sp) CRISPR/Cas9 system recognizes a total of 22 bases, twenty via the gRNA and two via the PAM NGG. Here we describe further development of the Neisseria meningitidis (Nm) orthologous CRISPR/Cas9 system for efficient and specific genome editing in mammalian cells. The Nm Cas9 recognizes a longer PAM sequence than that of Sp Cas9, which has the potential to result in fewer off-target sites genome-wide. The Nm CRISPR/Cas9 systems preferentially recognize longer target sites than the Sp system, and a longer PAM as previously identified, NNNNGATT. The Nm cleavage activity level is as high as 70%, similar to that of Sp Cas9. We tested the ability of Nm Cas9 to cleave targets with other PAM sequences and found that other PAM allowed cleavage, such that the PAM preference can be represented as NNNNGHTT. Interestingly, there was a difference between the one guide strand and two guide strand systems. The single guide strand system (sgRNA) displayed greater stringency in PAM recognition than the innate system (with separate tracrRNA and crRNA). Nm gRNAs were able to tolerate 1 base mismatches and 1 base DNA and RNA bulges to some extent when tested in a single-strand annealing assay. To further investigate the specificity of Nm CRISPR/Cas9 at the genomic level we used deep sequencing of potential off-target sites after CRISPR transfection. To directly compare the specificity of both Sp and Nm CRISPR/Cas9 systems, overlapping Nm and Sp target sites were identified in seven different genes. For the three targets where there was comparable on-target activity, potential Nm and Sp off-target sites were identified and subjected to deep sequencing using the Illumina MiSeq platform. We found that Sp CRISPR/Cas9 displayed greater levels of genome-wide off-target activity. In conclusion, our results demonstrate that the Nm CRISPR/Cas9 systems have better targeting specificity and thus provide an alternative to the widely used Sp CRISPR/Cas9.