Enhanced-specificity Streptococcus pyogenes Cas9 (eSpCas9)

Enhanced-specificity variant of Streptococcus pyogenes Cas9 carrying alanine substitutions in the protein groove that binds the nontarget DNA strand. eSpCas9 cleaves the same NGG PAM sites as wild-type SpCas9 with a 20-nucleotide guide, but tolerates far fewer guide-target mismatches and so produces less off-target editing in human cells.

Length: 4269 bp (1423 aa)

PAM: NGG

Cut type: blunt

Effector domain: RuvC and HNH

Origin: Engineered from Streptococcus pyogenes Cas9 by alanine substitution

Characteristics

This eSpCas9 carries K810A/K1003A/R1060A and another variant eSpCas9(1.1) carries K848A/K1003A/R1060A, all in the groove between the RuvC and HNH domains that holds the nontarget DNA strand. The substitutions are proposed to weaken nontarget-strand binding so the two DNA strands rehybridize unless the guide matches the target strand closely; single-molecule FRET shows eSpCas9(1.1) bound to a mismatched target is trapped in a catalytically inactive state. Both variants give indel levels similar to wild-type SpCas9 at most of 24 target sites across 10 human genomic loci, and reduce cleavage to under 0.2% indels at 22 of 24 tested off-target sites with 20-nucleotide guides in HEK cells. Guide length, NGG PAM requirement and blunt cleavage are unchanged from SpCas9.

Applications: Plasmid-delivered knockout editing in human cell lines where off-target cleavage must be minimized, validated at 24 target sites across 10 loci including EMX1, VEGFA, DNMT1, TPCN2, NRG2, CACNA2D4 and ADAMTSL1 in HEK cells. Mismatch discrimination extends to guides carrying a single mismatch outside the 7-12 base pair seed region, where wild-type SpCas9 still edits, so eSpCas9 fits sites with close sequence relatives in the genome.

Limitations: eSpCas9 needs a perfectly matched 20-nucleotide spacer. Extra 5' nucleotides, such as the G added by many U6 expression constructs, sharply reduce on-target activity. On-target activity also falls at some individual target sites, and no single high-fidelity variant is best across all targets, so eSpCas9 and SpCas9-HF1 must be matched to the site. The NGG PAM requirement is unchanged from SpCas9.

Sequence

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References

  1. Slaymaker et al. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science - Slaymaker 2016 eSpCas9
  2. Kleinstiver et al. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature - Kleinstiver 2016 SpCas9-HF1
  3. Chen et al. (2017). Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature - Chen 2017 HypaCas9
  4. Kulcsár et al. (2017). Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol - Kulcsár 2017 HeFSpCas9
  5. Zhang et al. (2017). Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases. Genome Biol - Zhang 2017 Matched Guides

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