High-fidelity variant of Streptococcus pyogenes Cas9 carrying four alanine substitutions that remove non-specific contacts to the target DNA backbone, which reduces off-target cleavage while leaving NGG PAM recognition and guide-directed cleavage intact. Used where genome-wide editing specificity matters more than maximum on-target activity.
Length: 4215 bp (1405 aa)
PAM: NGG
Cut type: blunt
Effector domain: RuvC and HNH
Origin: Engineered from Streptococcus pyogenes Cas9 (N497A/R661A/Q695A/Q926A)
Characteristics
Carries the substitutions N497A, R661A, Q695A and Q926A, which remove hydrogen bonds that wild-type SpCas9 makes to the phosphate backbone of the target DNA strand; with less non-specific binding energy available, cleavage becomes dependent on near-complete guide-target complementarity. The cleavage chemistry is unchanged, with the RuvC and HNH domains producing a blunt double-strand break three base pairs upstream of the NGG PAM. In U2OS cells, 32 of 37 sgRNAs retain at least 70% of wild-type SpCas9 on-target activity, most falling between 90% and 140%. GUIDE-seq detects no off-target sites for six of seven sgRNAs that produce off-targets with wild-type SpCas9, and 123 of 144 off-targets are eliminated at a repetitive VEGFA target.
Applications: Knockout generation at endogenous human loci including EMX1, FANCF, RUNX1, ZSCAN2 and VEGFA in U2OS cells, and editing in HEK293, HeLa and mouse N2a cells. Suited to targets that have closely homologous sites elsewhere in the genome, including repetitive and homopolymeric sequences. The four substitutions also transfer to the PAM-altered SpCas9-VQR and SpCas9-VRQR backbones, giving high-fidelity nucleases with shifted PAM requirements.
Limitations: On-target activity is lower than wild-type SpCas9 at many sites, with only 41% of targets tested reaching 80% of wild-type activity, against 83% for eSpCas9(1.1). Activity falls to roughly 16% of wild-type when the sgRNA carries an extra 5' G added for U6 transcription, so 21-nucleotide guides must be avoided. Truncated sgRNAs also impair on-target activity.
Kleinstiver et al. (2016).
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Nature - Kleinstiver 2016 SpCas9-HF1
Slaymaker et al. (2016).
Rationally engineered Cas9 nucleases with improved specificity.
Science - Slaymaker 2016 eSpCas9
Kulcsár et al. (2017).
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.
Genome Biol - Kulcsár 2017 HeFSpCas9
Kulcsár et al. (2020).
Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs.
Nat Commun - Kulcsár 2020 Blackjack
