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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

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.
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f5: 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.

Mentions: To test whether the proxy-CRISPR strategy can mediate CjCas9 target access, we used chromatin immunoprecipitation (ChIP) and droplet digital PCR (ddPCR) to measure the binding of a FLAG-tagged, catalytically dead CjCas9 (CjdCas9) with D8A and H559A mutations on the cleavable AAVS1 target and the uncleavable POR target from the guide length comparison in K562 cells, using the 20-nt guides (Fig. 5a,b). Indeed, although CjdCas9 was unable to bind the POR target on its own, the binding of SpdCas9 at two proximal locations enabled CjdCas9 to bind the otherwise inaccessible POR target even more efficiently than its binding to the accessible AAVS1 target by itself (Fig. 5c). To evaluate the efficacy of this strategy on CjCas9 target DNA cleavage, we used CjCas9 to edit three POR targets in K562 cells, including the target in the guide length comparison, using the 20-nt guides. While no cleavage was observed on any of the targets by CjCas9 alone, high cleavage efficiencies with indels ranging from 25.5 to 37.9% in two targets and from 5.1 to 16.5% in one target were achieved when CjCas9 was assisted by SpdCas9 binding at various proximal locations (Fig. 5d). It is worth noting that all three POR targets contain the 5′-NNNNACAY-3′ PAM, and we were unable to restore the CjCas9 nuclease activity on a different POR target bearing the 5′-NNNNACAA-3′ PAM, even with five different combinations of SpdCas9 proximal binding sites. This further confirmed the preference for the 5′-NNNNACAY-3′ PAM by CjCas9 in mammalian cells. In addition, we also used CjCas9 to edit one of the POR targets in HEK293 cells using the 20-, 22- and 24-nt guides. The editing efficiencies ranged from 19.8 to 24.4% indels when CjCas9 was aided by SpdCas9, whereas the efficiencies by CjCas9 alone were below detection levels (Supplementary Fig. 17). By using a single-stranded DNA oligo donor as DSB repair template, we verified that DSBs induced by the proxy-CRISPR strategy are amenable to homology-directed repair-mediated gene-editing applications (Supplementary Fig. 18).


Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting
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.
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f5: 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.
Mentions: To test whether the proxy-CRISPR strategy can mediate CjCas9 target access, we used chromatin immunoprecipitation (ChIP) and droplet digital PCR (ddPCR) to measure the binding of a FLAG-tagged, catalytically dead CjCas9 (CjdCas9) with D8A and H559A mutations on the cleavable AAVS1 target and the uncleavable POR target from the guide length comparison in K562 cells, using the 20-nt guides (Fig. 5a,b). Indeed, although CjdCas9 was unable to bind the POR target on its own, the binding of SpdCas9 at two proximal locations enabled CjdCas9 to bind the otherwise inaccessible POR target even more efficiently than its binding to the accessible AAVS1 target by itself (Fig. 5c). To evaluate the efficacy of this strategy on CjCas9 target DNA cleavage, we used CjCas9 to edit three POR targets in K562 cells, including the target in the guide length comparison, using the 20-nt guides. While no cleavage was observed on any of the targets by CjCas9 alone, high cleavage efficiencies with indels ranging from 25.5 to 37.9% in two targets and from 5.1 to 16.5% in one target were achieved when CjCas9 was assisted by SpdCas9 binding at various proximal locations (Fig. 5d). It is worth noting that all three POR targets contain the 5′-NNNNACAY-3′ PAM, and we were unable to restore the CjCas9 nuclease activity on a different POR target bearing the 5′-NNNNACAA-3′ PAM, even with five different combinations of SpdCas9 proximal binding sites. This further confirmed the preference for the 5′-NNNNACAY-3′ PAM by CjCas9 in mammalian cells. In addition, we also used CjCas9 to edit one of the POR targets in HEK293 cells using the 20-, 22- and 24-nt guides. The editing efficiencies ranged from 19.8 to 24.4% indels when CjCas9 was aided by SpdCas9, whereas the efficiencies by CjCas9 alone were below detection levels (Supplementary Fig. 17). By using a single-stranded DNA oligo donor as DSB repair template, we verified that DSBs induced by the proxy-CRISPR strategy are amenable to homology-directed repair-mediated gene-editing applications (Supplementary Fig. 18).

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