Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

Abstract

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

Figure 1. The type II-B FnCas9 from Francisella novicida cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs.

(a) Schematics of FnCas9 and SpCas9. BH, bridge helix; CTD, C-terminal domain; HNH, HNH nuclease domain; NLS, nuclear localization signal; RuvC-I-III, RuvC nuclease domain; REC, recognition lobe. (b) An EMX1 target on the purified DNA substrate used for cell-free cleavage assays. The protospacer is highlighted in purple and the PAM is underlined. The substrate was prepared by PCR from K562 genomic DNA. Blue triangles indicate cleavage positions by FnCas9 or SpCas9. (c) Run-off DNA sequencing on FnCas9 and SpCas9 cell-free cleavage products. The sequencing reads from a reverse primer show that FnCas9 cleaved the non-target strand 3–4 bp farther away from the PAM compared with the SpCas9 cleavage position. The sequencing reads from a forward primer show that both Cas9 nucleases cleaved the target strand at the same position. (d) Competitive ligation assays. dsDNA oligo inserts with compatible 3-nt or 4-nt 5′-overhangs without a 5′-phosphate group were ligated with FnCas9 or SpCas9 digested plasmid vectors. Inserts with 4-nt 5′-overhangs were predominant (87%) in recombinant plasmid DNA.

Figure 2. Single-nucleotide specificity comparison between FnCas9 and SpCas9.

(a) An EMX1 target and the corresponding wild-type guide sequence for the specificity comparison in human K562 cells. The protospacer is highlighted in purple and the PAM is underlined. The wild-type guide sequence was sequentially mutated at each position with three different types of mismatch. The wild-type guide sequence and single-mismatched guide sequences were each cloned into the FnCas9 and SpCas9 sgRNA plasmid vectors, respectively. (b) Cleavage activities (% indels) of FnCas9 and SpCas9 with the wild-type guide sequence (n=3, error bar shows mean±s.d.). (c) Cleavage activities (% indels) of FnCas9 and SpCas9 with type I single-mismatched guide sequences that were designed to create the most disruptive effect on base paring between an RNA base and a DNA base (rA:dA, rA:dG, rC:dC and rC:dT) (n=3, error bar shows mean±s.d.). (d,e) Cleavage activities (% indels) of FnCas9 and SpCas9 with type II and III single-mismatched guide sequences that were designed to create a less disruptive effect on base paring between an RNA base and a DNA base (rG:dA, rU:dG, rU:dC, rU:dT; and rC:dA, rG:dG, rA:dC, rG:dT; n=3, error bar shows mean±s.d.).

Figure 3. Variation of FnCas9 and SpCas9 nuclease activity on human chromosomal DNA.

FnCas9 and SpCas9 were respectively targeted to the same genomic sites in K562 cells and the cleavage activities were measured by Surveyor Nuclease S digestion assays. Target positions were plotted based on the PAM positions on either the sense or antisense strand. Target sequences are listed in Supplementary Data 1. (a) Nuclease activities (% indels) of FnCas9 and SpCas9 in the human CAR locus on chromosome 1. (b) Nuclease activities (% indels) of FnCas9 and SpCas9 in the human POR locus on chromosome 7.

Figure 4. Restoration of FnCas9 nuclease activity in K562 cells by catalytically dead SpCas9 (SpdCas9) binding at proximal locations.

(a) Schematic of a proximity chromatin interference hypothesis. FnCas9 is unable to access an endogenous target in a certain chromatin configuration, but the binding of SpdCas9 at proximal locations alters the local chromatin homeostasis and enables FnCas9 to access and cleave the otherwise inaccessible target. (b) FnCas9 and SpdCas9 targets in the POR exon 8 genomic region for testing the hypothesis. Targets are indicated by bars and PAMs are highlighted in purple. FnCas9 cleavage positions are indicated by yellow triangles. (c) FnCas9 target DNA cleavage activities under different conditions. The binding of SpdCas9 at one proximal site restored FnCas9 cleavage activity and two SpdCas9 proximal binding sites acted synergistically. Data are representative of three independent experiments. The sgRNA numbers correspond to the target numbers in b. M, wide-range DNA markers; ND, not determined.

Figure 5. Analysis of target binding and cleavage by the type II-C Cas9 from Campylobacter jejuni (CjCas9) in K562 cells.

(a) CjCas9 and SpdCas9 targets in the human POR and AAVS1 loci. The AAVS1 target had previously been determined to be cleavable by CjCas9, while the three CjCas9 targets in POR had previously been determined to be uncleavable in K562 cells. Targets are indicated by bars and PAMs are highlighted in purple (SpdCas9) and dark blue (CjCas9). (b) Schematic of target binding assays by chromatin immunoprecipitation (ChIP) and droplet digital PCR (ddPCR). CjCas9 was converted to a catalytically dead Cas9 (CjdCas9) with D8A and H559A double mutations and tagged at the N terminus with a 3XFLAG epitope (FLAG-CjdCas9). (c) FLAG-CjdCas9 target binding activities on the AAVS1 target and POR target 1 with or without the assistance of SpdCas9 binding at proximal locations. The sgRNA numbers correspond to the target numbers in a (n=3 biological replicates; error bars show mean±s.d.). (d) CjCas9 cleavage activities (% indels) on the three POR targets with or without the assistance of SpdCas9. The sgRNA numbers correspond to the target numbers in a. Data are representative of three independent experiments. M, wide-range DNA markers; ND, not determined.

Figure 6. SpdCas9-assisted gene editing by the type II-C Cas9 from Neisseria cinerea (NcCas9).

(a) NcCas9 and SpdCas9 targets in the human POR locus. Targets are indicated by bars and PAMs are highlighted in purple (SpdCas9) and dark blue (NcCas9). (b) NcCas9 target cleavage activities (% indels) with or without the assistance of SpdCas9 binding at proximal locations. The sgRNA numbers correspond to the target numbers in a. M, wide-range DNA markers; ND, not determined.

Figure 7. SpdCas9-assisted gene editing by the type V Cpf1 from Francisella novicida (FnCpf1) in K562 cells.

(a) FnCpf1 and SpdCas9 targets in the human POR locus. Targets are indicated by bars and PAMs are highlighted in purple (SpdCas9) and dark blue (FnCpf1). (b) FnCpf1 target cleavage activities (% indels) with or without SpdCas9 binding at proximal locations. The Fn-crRNA and Sp-sgRNA numbers correspond to the target numbers in a. Data are representatives of three independent experiments. M, wide-range DNA markers; ND, not determined.

Figure 8. Selective editing on identical targets in human HBB and HBD by proxy-CRISPR strategy.

(a) CjCas9 and SpdCas9 targets in the human haemoglobin subunit beta (HBB) and subunit delta (HBD) loci. Targets are indicated by bars and PAMs are highlighted in purple (SpdCas9) and dark blue (CjCas9). The two identical CjCas9 targets in HBB and HBD are highlighted by a rectangle. (b) CjCas9 cleavage activities on the HBB and HBD identical targets in different combinations with SpdCas9 and Sp-sgRNAs. CjCas9 selectively cleaved the HBB target when it was co-expressed with SpdCas9 and a pair of Sp-sgRNAs specific to two proximal sites in HBB. Conversely, CjCas9 selectively cleaved the HBD target when it was co-expressed with SpdCas9 and a pair of Sp-gRNAs specific to two proximal sites in HBD. The sgRNA numbers correspond to the target numbers in a. The two digested bands on the first two lanes of the right panel and the first three lanes of the left panel were derived from SNPs in K562 cells and were excluded from cleavage determination. Data are representatives of three independent experiments. M, wide-range DNA markers; ND, not determined.