Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Abstract

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue

“…The closer to the PAM sequence a mismatch is, the less likely it is to be edited. Therefore, raising stringency and specificity, eliminating off-target editing by CRISPR/Cas9 technologies through molecular engineering, and/or discovering novel Cas9-like genes from eubacteria or archaea, such as a high specificity FnCas9 from the bacterium Francisella novicida [32], will be the focus for future gene editing research before CRISPR technology could be adopted for human genetic and medical applications.…”

Rapid, field-deployable nucleobase detection and identification using FnCas9

“…DNA samples were directly PCR amplified for the target region while RNA samples were converted to cDNA using Reverse Transcriptase kit (Qiagen) and PCR amplified. In vitro cleavage assay was performed as optimized in our previous study 16…”

“…Chimeric gRNA (crRNA:TracrRNA) was prepared by mixing in-vitro transcribed crRNA and synthetic 3'-FAM labelled TracrRNA in a equimolar ratio using annealing buffer (100mM NaCl, 50mM Tris-HCl pH 8 and 1mM MgCl 2 ), mix was heated at 95 C for 2-5 minutes and then allowed to cool at room temperature for [15][16][17][18][19][20] minutes. Chimeric gRNA-dead FnCas9 RNP complex (500nM) was prepared by mixing them in a buffer (20mM HEPES, pH7.5, 150mM KCl, 1mM DTT, 10% glycerol, 10mM MgCl 2 ) and incubated for 10 min at RT.…”

“…We have recently reported a Cas9 ortholog from Francisella novicida (FnCas9) showing very high mismatch sensitivity both under in vitro and in vivo conditions [16][17][18] . This is based on its negligible binding affinity to substrates that harbor mismatches, a property that is distinct from engineered Cas proteins showing similar high specificity 19 .…”

TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

“…sgRNAs, 1′ and 1′Az, were designed to match a validated protospacer sequence used to target telomeric repeat in cells 52,53 . Nuclear puncta corresponding to telomeres were visualized for both 1′ and 1′Az, which is consistent with the telomere localization pattern established using fluorescent dCas9 fusion proteins (Figure 8a, see Figure 8b for single cell image) 12,24,53 . In a control experiment, non-targeting sgRNA 2Az showed fluorescence signal, which was non-specifically distributed all throughout the nucleus.…”