Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Abstract

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

Figure 1.

Functional PAM sequences for robust and potent DNA cleavage by St1Cas9 LMD9 in mammalian cells. (A) Schematic representations of St1Cas9 LMD9 flanked by nuclear localization signals (NLSs) and its engineered sgRNA (v1). Nucleotide sequence and functional modules are depicted; crRNA (green), loop (gray), tracrRNA (blue), and mutated nucleotides (orange). (B) K562 cells stably expressing St1Cas9 were transfected with indicated sgRNA expression vectors at increasing doses, and TIDE assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. The experiment was performed twice and yielded equivalent results; only one is shown. (C) Screening for guides targeting St1Cas9 LMD9 to various PAMs was performed by transient transfections in K562 (solid shapes) and Neuro-2a (open shapes) cells using single-vector constructs driving the expression of St1Cas9 and its sgRNA. Surveyor assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. See also Supplemental Figure S1.

Figure 2.

Structural basis for PAM specificity of engineered St1Cas9 variants with expanded targeting range. (A) Schematic representation of St1Cas9 hybrid proteins containing the N terminus of LMD9 and the C-terminal domains (WED + PI) of LMG18311 or CNRZ1066. To determine the activity of St1Cas9 variants programmed with sgRNAs compatible with different PAMs, K562 cells were transiently transfected with single-vector constructs driving expression of St1Cas9 and its sgRNA. For each PAM and nuclease combination, four different sgRNAs (targets) were tested. Surveyor assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. The experiment was performed twice and yielded equivalent results; only one is shown. (B) Close-up view of the 5′-GCAGAAA-3′ PAM bound to the St1Cas9 (DGCC7010) PI domain (PDB: 6RJD). The target (turquoise) and nontarget (blue) strands are shown as sticks (the phosphate–sugar backbones are also shown as ribbons). The ribbon representation of the PI domain is orange. The hydrogen bonds between the side chain of St1Cas9 K1086 and the nucleobase of dG4 is shown as a dashed line. (C,D) The PI domains of St1Cas9 and SaCas9 (PDB: 5CZZ, gray ribbon) are superimposed. (C) The St1Cas9 Q1084 and SaCas9 R1015 occupy the same positions relative to the PAM (dA3). The St1Cas9 E1057 and SaCas9 E993 occupy the same positions relative to St1Cas9 Q1084 and SaCas9 R1015, respectively. (D) St1Cas9 T1048 and M1049 (substituted for N1048 and D1049 in some variants) superimpose onto SaCas9 N985 and N986 that specify purines in positions 4 and 5 of the PAM. See also Supplemental Figure S3.

Figure 3.

Broadening the targeting scope of base editors using St1Cas9 variants. (A) K562 cells were transiently transfected with single-vector constructs driving expression of St1BE4max LMD9 and its sgRNA. Genomic DNA was harvested 3 d later, and quantification of base editing was performed on PCR amplified target sites using EditR. The target sequence was defined as the 20 bases upstream of the PAM and are numbered in decreasing order from the PAM. Sequence of the guides and related PAMs are shown with target cytosine highlighted in blue. An expression vector encoding EGFP (−) was used as a negative control. (B–D) Same as A but using St1BE4max LMG18311, CNRZ1066, and TH1477 chimeric proteins. (E) Same as A but using St1ABEmax LMD9. Target adenines are highlighted in red. Most sgRNAs were tested at least twice; only one experiment is shown. See also Supplemental Figure S5.

Figure 4.

In vivo genome editing using St1Cas9. (A) The tyrosine degradation pathway and associated inborn errors of metabolism (IEMs). (B) Experimental design. Neonatal (2-d-old) Fah^−/− mice were injected with AAV8-St1Cas9 or saline into the retro-orbital sinus and weaned at 21 d, and NTBC was removed at 30 d of age. Mice off NTBC were sacrificed when they lost 20% of their body weight. (C) Schematic representation of the AAV-St1Cas9 v3 vector. Annotated are the liver-specific promoter (LP1b), synthetic polyadenylation sequence (SpA), and hU6 promoter. Arrows indicate the direction of transcriptional unit. (D) Neonatal Fah^−/− mice were injected with either 5 × 10¹⁰ or 1 × 10¹¹ vector genomes (vg) of AAV8-St1Cas9 v3 targeting Hpd exon 13 and sacrificed 28 d following injection or kept alive for phenotypic and metabolic studies for 4 mo post NTBC removal. Genomic DNA was extracted from whole-liver samples, and the Surveyor assay was used to determine the frequency of indels. Each dot represents a different mouse. A mouse injected with saline (−) was used as a negative control. (E) SUAC levels in urine from treated mice were determined 15 d (short term) or 4 mo (long term) following NTBC removal. Samples were collected from the indicated treatment groups over a 24-h period using metabolic cages. Number of mice per group/metabolic cage (n) and AAV doses (vg) is indicated. SUAC levels are undetectable in C57BL/6N (wild-type) mice. (F–H) Survival analysis, body weight, and glycemia following NTBC removal in treated mice. Body weight was measured daily, and glycemia was monitored in nonfasted mice. Solid lines designate the mean; and error bars are represented by shaded areas and denote SEM. See also Supplemental Figure S6.