Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification

Abstract

Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ~15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 ‘nickases’, recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least 4-fold higher_than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the broad versatility of this approach for highly specific genome-wide editing.

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*

“…The introduction of two sgRNAs as described in our deletion schema theoretically carries twice the frequency of off-target mutations as compared with a single sgRNA. Off-target effects may be minimized using a double nickase strategy for DSBs (6,22), truncated sgRNAs (32), or dimeric Cas9-FokI fusions (33,34), but it remains to be determined how these approaches might affect deletion frequency. Complementation of the deletion phenotype by reintroduction of the deleted sequence would verify the association of the deletion to the phenotype but may be laborious to achieve.…”

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

“…To minimize or avoid RGEN off-target effects, we and others have proposed various methods, which include dimeric Cas9 systems (paired Cas9 nickases [Mali et al 2013a;Ran et al 2013;Cho et al 2014] and dCas9-FokI [Guilinger et al 2014;Tsai et al 2014]), delivery of RGEN ribonucleoproteins (RNPs) Ramakrishna et al 2014;Zuris et al 2015), and modified guide RNAs Fu et al 2014). The dimeric systems require two active sgRNAs and two adjacent PAMs, limiting targetable sites.…”

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…However, due to the limited sites we analysed and the restricted sensitivity of the T7EI cleavage assay, lower incidents of OTEs for these sgRNAs at a genome-wide scale remain to be determined by more powerful approaches, such as ultradeep next-generation sequencing of the whole genome Smith et al, 2014;Veres et al, 2014). Studies aimed to develop modified CRISPR/Cas9 systems are also promising to further improve the on-target fidelity of CRISPR/Cas9 (Guilinger et al, 2014;Qi et al, 2013;Ran et al, 2013).…”