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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

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.
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f8: 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.

Mentions: We reasoned that the proxy-CRISPR targeting strategy can be applied to reduce off-target effects. For a majority of targets, DSBs by an inactive CRISPR–Cas nuclease will require at least two guide RNA binding sites proximal to each other on a chromosome. The likelihood of two similar genomic sites occurring elsewhere in the genome greatly diminishes compared to one site. To test this assumption, we examined potential off-target sites corresponding to the POR target site (Fig. 4b) and the two EMX1 target sites (Supplementary Fig. 13) that were edited by FnCas9 with the assistance of SpdCas9 proximal binding. In all instances, we did not find similar genomic sites corresponding to those SpdCas9 proximal binding sites in the POR and EMX1 loci and no off-target cleavage by FnCas9 was detected on any of these potential off-target sites (Supplementary Table 1). We further postulated that identical genomic sites in different genes can be selectively modified using this strategy by judiciously selecting proximal dCas9-binding sites to differentiate the identical genomic sites. To demonstrate a proof of concept on this application, we used CjCas9 in concert with SpdCas9 to selectively edit two identical targets in the human haemoglobin subunit beta (HBB) and subunit delta (HBD) loci (Fig. 8a). When expressed alone, CjCas9 was unable to cleave either the HBB target or the HBD target in K562 cells. But when expressed in combination with SpdCas9 and a pair of sgRNAs specific to two proximal binding sites in HBB, CjCas9 cleaved the HBB target efficiently (32% indels) without cleaving the identical target in HBD (Fig. 8b). Conversely, CjCas9 was able to selectively cleave the HBD target fairly efficiently (14% indels) without cleaving the identical target in HBB when it was aided by SpdCas9 and a pair of sgRNAs specific to two proximal binding sites in HBD (Fig. 8b). No off-target cleavage by CjCas9 was detected on four potential off-target sites (Supplementary Table 1). These results demonstrate the potential of this strategy for precision gene-editing applications.


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

License
Show All Figures
getmorefigures.php?uid=PMC5385574&req=5

f8: 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.
Mentions: We reasoned that the proxy-CRISPR targeting strategy can be applied to reduce off-target effects. For a majority of targets, DSBs by an inactive CRISPR–Cas nuclease will require at least two guide RNA binding sites proximal to each other on a chromosome. The likelihood of two similar genomic sites occurring elsewhere in the genome greatly diminishes compared to one site. To test this assumption, we examined potential off-target sites corresponding to the POR target site (Fig. 4b) and the two EMX1 target sites (Supplementary Fig. 13) that were edited by FnCas9 with the assistance of SpdCas9 proximal binding. In all instances, we did not find similar genomic sites corresponding to those SpdCas9 proximal binding sites in the POR and EMX1 loci and no off-target cleavage by FnCas9 was detected on any of these potential off-target sites (Supplementary Table 1). We further postulated that identical genomic sites in different genes can be selectively modified using this strategy by judiciously selecting proximal dCas9-binding sites to differentiate the identical genomic sites. To demonstrate a proof of concept on this application, we used CjCas9 in concert with SpdCas9 to selectively edit two identical targets in the human haemoglobin subunit beta (HBB) and subunit delta (HBD) loci (Fig. 8a). When expressed alone, CjCas9 was unable to cleave either the HBB target or the HBD target in K562 cells. But when expressed in combination with SpdCas9 and a pair of sgRNAs specific to two proximal binding sites in HBB, CjCas9 cleaved the HBB target efficiently (32% indels) without cleaving the identical target in HBD (Fig. 8b). Conversely, CjCas9 was able to selectively cleave the HBD target fairly efficiently (14% indels) without cleaving the identical target in HBB when it was aided by SpdCas9 and a pair of sgRNAs specific to two proximal binding sites in HBD (Fig. 8b). No off-target cleavage by CjCas9 was detected on four potential off-target sites (Supplementary Table 1). These results demonstrate the potential of this strategy for precision gene-editing applications.

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