FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity

Abstract

Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5′-NNTA-3′) and a guide RNA length of 21–22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.

Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering

“…In addition to SpCas9 and SaCas9, many other Cas9 proteins (e.g., ScCas9 [ 83 ], CjCas9 [ 84 ], NmeCas9 [ 85 ], FnCas9 [ 86 ], SmacCas9 [ 87 ], SauriCas9 [ 88 ], St1Cas9 [ 89 ], St3Cas9 [ 90 ], FrCas9 [ 34 ], to name a few) have been isolated and some of them were reported to be intrinsically high-fidelity [ 34 , 35 ]. Some of them were engineered with a broad PAM range, high fidelity, and/or both [ 91 ].…”

“…Instead, we focus on strategies and implementations reported to increase editing specificity. The strategies utilized for reducing off-target effects include Cas9 nuclease engineering [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], using natural Cas9 nucleases with high specificity [ 34 , 35 ], the utility of base editors [ 36 , 37 , 38 ] and prime editors [ 39 , 40 ], gRNA design optimization and/or modulation [ 8 , 9 , 41 , 42 , 43 , 44 , 45 , 46 ], control of Cas9′s activity via direct delivery (e.g., ribonucleoprotein (RNP) [ 20 , 47 ], virus-like particles [ 48 , 49 , 50 , 51 ], cell-penetrating peptide-mediated delivery [ 52 ], mRNA [ 53 ], and self-limiting circuits [ 54 ]), and combination with anti-CRISPR proteins or CRISPR inhibitors [ 55 , 56 , 57 , 58 ]. Among these strategies, direct or indirect engineering of Cas9 proteins represents the majority of efforts, evidenced by the relatively large number of studies toward this direction ( Table 1 ).…”

Plant_SNP_TATA_Z-Tester: A Web Service That Unequivocally Estimates the Impact of Proximal Promoter Mutations on Plant Gene Expression

“…Synthetic pinpoint optimization of plant gene promoters [ 3 ] to adapt plants to various environmental conditions during plant development (e.g., drought under climate change [ 4 ]) is becoming a part of the mainstream postgenomic agriculture progress [ 5 ] along with the design of hybrids of cultivated plants with their wild congeners [ 6 ] and both quantitative trait locus (QTL)- and single-nucleotide polymorphism (SNP) marker-assisted breeding [ 7 ]. Recently, a Faecalibaculum rodentium Cas9 protein for genome-editing CRISPR/Cas9 systems was found whose protospacer-adjacent motif (PAM) “NNTA” matches TATA-binding protein (TBP)-binding sites of eukaryotic promoters [ 8 ]. The ability of this protein to directly target the TATA box was confirmed for TATA-containing promoters of human genes ABCA1 , UCP1, and RANKL [ 8 ].…”

“…Recently, a Faecalibaculum rodentium Cas9 protein for genome-editing CRISPR/Cas9 systems was found whose protospacer-adjacent motif (PAM) “NNTA” matches TATA-binding protein (TBP)-binding sites of eukaryotic promoters [ 8 ]. The ability of this protein to directly target the TATA box was confirmed for TATA-containing promoters of human genes ABCA1 , UCP1, and RANKL [ 8 ].…”

Versatile and efficient genome editing with Neisseria cinerea Cas9

“…To date, a series of natural Cas9 orthologs and artificially-designed Cas9 variants with various PAM requirements have been developed, such as SaCas9 (NNGRRT) 9 , Nme1Cas9 (NNNNGATT) 10 , Nme2Cas9 (NNNNCC) 11 , St1Cas9 (NNRGAA) 12 , SpaCas9 (NNGYRA) 13 , Cje1Cas9 (NNNVRYAC) 14 , Cje3Cas9 (NNNNCYA) 15 , SauriCas9 (NNGG) 16 , SchCas9 (NNGR) 17 , FrCas9 (NNTA) 18 , ScCas9 (NNG) 19 , SpCas9-VQR (NGA) 20 , SpCas9-NG (NG) 21 , SpCas9-NRNH (NRNH) 22 , SpG (NG) 23 , SpRY (NR > NY) 23 , etc. These Cas9 nucleases expand the PAM compatibility of Cas9 orthologs and improve the flexibility of genome editing applications 24 .…”