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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f1: 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.

Mentions: We developed a human codon optimized type II-B FnCas9 nuclease from Francisella novicida U112 (Fig. 1a; Supplementary Fig. 1) and an FnCas9-specific sgRNA scaffold (Supplementary Fig. 2) for genome-editing exploration. By surveying a large number of endogenous targets in human K562 cells, we determined that the FnCas9 in vivo PAM specificity (Supplementary Fig. 3) is consistent with the previous biochemical data showing that the FnCas9 PAM is 5′-NGG-3′, with the preference for an H (A, T or C) in the first PAM position37. In comparing FnCas9 with SpCas9 on the same endogenous targets, we noticed a subtle difference between these two nucleases in the acrylamide gel mobility of edited DNA fragments after Surveyor Nuclease S digestion. We then sequenced an EMX1 (empty spiracles homeobox 1) target and a POR (cytochrome p450 oxidoreductase) target and observed that the FnCas9-mediated mutations were frequently initiated at positions farther upstream from the PAM in comparison with the SpCas9-mediated mutations. By conducting run-off DNA sequencing on cell-free cleavage fragments of the EMX1 target (Fig. 1b), we found that, unlike SpCas9, FnCas9 indeed cleaved the non-target strand at 6–7 bp from the PAM (Fig. 1c), whereas both Cas9 nucleases cleaved the target strand at 3 bp from the PAM, as previously reported5. This staggered cleavage feature by FnCas9 has not been found in other Cas9 nucleases, but is consistent with the recently published FnCas9 crystal structure data37. In contrast to SpCas9 and SaCas9, the RuvC nuclease domain of FnCas9 does not interact with the PAM-interacting C-terminal domain and is positioned farther away from the PAM37. This structural configuration substantiates the staggered cleavage functionality of FnCas9. To further define the cleavage pattern, we performed a competitive DNA cloning assay by ligating FnCas9 digested plasmid DNA with non-phosphorylated double-stranded DNA (dsDNA) oligo inserts, carrying compatible 3-nt or 4-nt 5′-overhangs in an equal molar ratio. Colony sequencing revealed that 87% of recombinant clones contained the 4-nt 5′-overhang insert and 13% contained the 3-nt 5′-overhang insert (Fig. 1d). Thus, we conclude that FnCas9 predominantly cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs, each with a 5′-phosphate group.


Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting
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.
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Related In: Results  -  Collection

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f1: 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.
Mentions: We developed a human codon optimized type II-B FnCas9 nuclease from Francisella novicida U112 (Fig. 1a; Supplementary Fig. 1) and an FnCas9-specific sgRNA scaffold (Supplementary Fig. 2) for genome-editing exploration. By surveying a large number of endogenous targets in human K562 cells, we determined that the FnCas9 in vivo PAM specificity (Supplementary Fig. 3) is consistent with the previous biochemical data showing that the FnCas9 PAM is 5′-NGG-3′, with the preference for an H (A, T or C) in the first PAM position37. In comparing FnCas9 with SpCas9 on the same endogenous targets, we noticed a subtle difference between these two nucleases in the acrylamide gel mobility of edited DNA fragments after Surveyor Nuclease S digestion. We then sequenced an EMX1 (empty spiracles homeobox 1) target and a POR (cytochrome p450 oxidoreductase) target and observed that the FnCas9-mediated mutations were frequently initiated at positions farther upstream from the PAM in comparison with the SpCas9-mediated mutations. By conducting run-off DNA sequencing on cell-free cleavage fragments of the EMX1 target (Fig. 1b), we found that, unlike SpCas9, FnCas9 indeed cleaved the non-target strand at 6–7 bp from the PAM (Fig. 1c), whereas both Cas9 nucleases cleaved the target strand at 3 bp from the PAM, as previously reported5. This staggered cleavage feature by FnCas9 has not been found in other Cas9 nucleases, but is consistent with the recently published FnCas9 crystal structure data37. In contrast to SpCas9 and SaCas9, the RuvC nuclease domain of FnCas9 does not interact with the PAM-interacting C-terminal domain and is positioned farther away from the PAM37. This structural configuration substantiates the staggered cleavage functionality of FnCas9. To further define the cleavage pattern, we performed a competitive DNA cloning assay by ligating FnCas9 digested plasmid DNA with non-phosphorylated double-stranded DNA (dsDNA) oligo inserts, carrying compatible 3-nt or 4-nt 5′-overhangs in an equal molar ratio. Colony sequencing revealed that 87% of recombinant clones contained the 4-nt 5′-overhang insert and 13% contained the 3-nt 5′-overhang insert (Fig. 1d). Thus, we conclude that FnCas9 predominantly cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs, each with a 5′-phosphate group.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus