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Identification of SaCas9 orthologs containing a conserved serine residue that determines easy NNGG PAM recognition


The Clustered Frequently Interspaced Brief Palindromic Repeats (CRISPR)-RNA-guided Cas endonuclease system relies on the bacterial adaptive immune system and has been utilized as a quick and environment friendly technique for exact genome enhancing [16]. This method is made up of two principal parts: a Cas9 nuclease and a chimeric single-guide RNA (sgRNA) derived from CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA) [2]. Cas9 and sgRNA mix to type a fancy that acknowledges the goal DNA that’s complementary to the 5′ finish of the sgRNA [2]. Along with sgRNA-target DNA complementarity, DNA recognition requires a particular DNA sequence often known as protospacer adjoining motif (PAM), flanking the goal sequence [2]. The PAM permits the Cas nuclease to discriminate between the goal DNA and the DNA sequence encoding the sgRNA but in addition restricts its capacity to focus on any sequence within the genome.

Enhancing effectivity is a significant hurdle of the CRISPR system. Each Cas nuclease has its personal nucleotide choice [7]. For instance, SpCas9 prefers guanine-rich sequences [8], whereas AsCas12a prefers adenine-rich sequences [9]. SpCas9 is mostly thought-about probably the most environment friendly Cas nuclease, whose effectivity varies from 0% to roughly 100% relying on the goal sequences [8]. Though earlier research have centered on limitations of the PAM [1012], the only real presence of a PAM inside a locus doesn’t assure that it may be effectively edited. For top effectivity of genome enhancing to be achieved, it’s important to ascertain a Cas9 repertoire that may accommodate all sequences.

Cas9 nucleases with versatile PAM necessities are essential for large-scale genome enhancing. We beforehand developed Cas9 nucleases with extremely versatile NNGG PAMs recognition [13,14]. To quickly establish extra pure Cas9 nucleases recognizing NNGG PAMs, we in contrast the PAM-interacting (PI) domains of SaCas9 orthologs and located that the serine residue similar to SaCas9 N986 was related to the NNGG PAM. We recognized 5 extra SaCas9 orthologs recognizing the NNGG PAM. We additional engineered two of them to enhance the specificity. Our examine expands the Cas9 repertoire and offers a basis to seek for Cas9s with NNGG PAMs sooner or later.


A serine residue was related to the NNGG PAM requirement amongst SaCas9 orthologs

We beforehand recognized PAMs for 16 SaCas9 orthologs, the place SauriCas9 and SlugCas9 acknowledged NNGG PAMs [1315]. Nishimasu and colleagues have demonstrated that amino acids of N985, N986, R991, E993, and R1015 within the PI area of SaCas9 are essential for PAM recognition [16]. N985, E993, and R1015 are very conserved amongst these 16 orthologs (Fig 1A). In distinction, residues similar to N986 and R991 confirmed substantial range. Apparently, SauriCas9 and SlugCas9 include a serine residue similar to SaCas9 N986. We hypothesized that this serine residue is related to the NNGG PAM.


Fig 1. Evaluation of 5 SaCas9 ortholog actions.

(A) Amino acid sequences of the SaCas9 ortholog PI area are aligned. The residues which can be necessary for PAM recognition are indicated on the high; the conserved residues amongst newly recognized SaCas9 orthologs are proven in pink; the names of newly recognized Cas9s are proven in inexperienced. (B) Design of the GFP activation reporter assemble. A goal sequence (protospacer) containing a 7-bp random sequence is inserted between ATG and the GFP-coding sequence. The library DNA is stably built-in into HEK293T cells by lentivirus. (C) Transfection of SaCas9 orthologs induced GFP expression. Share of GFP-positive cells was proven. The cells with out transfection of Cas9 have been used as a detrimental management.


We employed SaCas9 as a template to seek for associated orthologs from NCBI’s Gene database, and our search recognized 5 extra Cas9s that contained this serine residue with amino acid identification starting from 58.4% to 64.5% (Fig 1A and Desk 1). Genetic loci of those orthologs include a conserved group the place Cas9 is adopted by Cas1 and Cas2 (S1A Fig). The group of CRISPR repeats and tracrRNAs doesn’t seem like conserved. SaCas9 encodes a tracrRNA upstream of Cas9 and CRISPR repeats downstream of Cas2. Sha2Cas9 and SpeCas9 encode CRISPR repeats and tracrRNAs downstream of Cas2. SwaCas9 and Swa2Cas9 encode CRISPR repeats and tracrRNAs upstream of Cas9 and extra CRISPR repeats downstream of Cas2. SmiCas9 encodes CRISPR repeats-tracrRNA-CRISPR repeats upstream of Cas9.

Nonetheless, the 5′ finish sequences of CRISPR repeats and tracrRNAs exhibited excessive conservation amongst these orthologs (S1B and S1C Fig). We fused the three′ finish of a direct repeat with the 5′ finish of the corresponding tracrRNA, together with the full-length tail, through a 4-nt linker to type a sgRNA for every Cas9 (S2A Fig). Apparently, these sgRNAs fashioned the same secondary buildings with three stem loops (S2B Fig), suggesting that these SaCas9 orthologs might share the identical sgRNA scaffold for genome enhancing.

PAM screening

Subsequent, we used a beforehand established GFP activation assay for PAM screening [13,15]. On this assay, the GFP expression is disrupted by a goal sequence (protospacer) flanked by a 7-bp random sequence, which is inserted into the GFP coding sequence instantly downstream of the ATG begin codon, inducing to a frameshift mutation. This reporter library is then stably built-in into HEK293T cells. If a Cas9 nuclease efficiently edits the goal sequence, small insertions or deletions (indels) can be generated on the goal sequence, and a useful GFP cassette can be restored in a portion of cells (Fig 1B). Every Cas9 was human codon optimized, synthesized, and cloned right into a mammalian expression assemble that was developed by Ran and colleagues [17]. The canonical SaCas9 sgRNA scaffold was employed for sgRNA expression [17]. Three days after transfection of Cas9 with sgRNA expression plasmids, all 5 examined Cas9s induced GFP expression (Fig 1C). GFP-positive cells have been sorted out and the goal DNA was PCR amplified for deep sequencing. Sequencing outcomes confirmed that indels occurred at goal websites (Fig 2A). WebLogos and PAM wheels have been generated primarily based on deep sequencing information, which revealed that these Cas9s acknowledged NNGG PAMs (Fig 2B and 2C). These information validated our speculation that the serine residue is related to the NNGG PAM.


Fig 2. Evaluation of the PAM sequence of Cas9.

(A) Deep sequencing reveals that SmiCas9, Sha2Cas9, and SpeCas9 generated indels on the targets. (B) WebLogos have been generated primarily based on the deep sequencing information. (C) PAM wheels have been generated primarily based on the deep sequencing information.


To check whether or not the serine residue similar to SaCas9 N986 decided NNGG PAM recognition, we changed N986 with a serine (S3A Fig). The GFP activation assay revealed that the substitution elevated guanine choice at PAM place 3, however the favored PAM remained NNGRRT (S3B Fig). We reanalyzed the PI area of those orthologs and recognized two extra residues, a glutamic acid (E) similar to SaCas9 P1013 and a lysine (Ok) similar to SaCas9 I1017, conserved amongst orthologs that acknowledged an NNGG PAM, besides SmiCas9 the place there’s a 13-amino acid insertion similar to SaCas9 P1013 (Fig 1A). We added both P1013E, I1017K, or each mutations to SaCas9-N986 (S3A Fig). Apparently, all resulted SaCas9 variants acknowledged an NNGG PAM (S3B Fig). These information demonstrated that these three amino acids are necessary for figuring out the NNGG PAM.

Genome enhancing for endogenous loci

Subsequent, we examined the capability of those Cas9s for genome enhancing at chosen endogenous websites in HEK293T cells. 5 days after transfection of Cas9 and sgRNA expression plasmid DNA, we extracted genomic DNA and amplified goal websites by PCR. As an preliminary display, we used the T7EI assay to quickly analyze the effectivity for every Cas9. SmiCas9, Sha2Cas9, and SpeCas9 displayed increased enhancing effectivity, whereas SwaCas9 and Swa2Cas9 displayed decrease enhancing effectivity (S4A and S4B Fig). Within the subsequent experiments, we solely centered on SmiCas9, Sha2Cas9, and SpeCas9.

We in contrast the exercise of those three Cas9s to that of SaCas9 at 13 endogenous websites with NNGGRT PAMs. All examined Cas9s have been expressed from the identical assemble and achieved comparable expression ranges, as revealed by western blot (Fig 3A and 3B). All 4 Cas9 nucleases generated indels with completely different efficiencies relying on the goal websites in HEK293T cells (Fig 3C). Apparently, these Cas9s displayed completely different actions at some websites. For instance, Sha2Cas9 displayed increased exercise at website E0, whereas SmiCas9 and SpeCas9 displayed increased exercise at website G10. SaCas9 displayed decrease effectivity than newly recognized Cas9s at websites G3 and G9. These information demonstrated that these Cas9s choose distinct goal sequences. Total, SaCas9, Sha2Cas9, and SpeCas9 displayed comparable actions, whereas SmiCas9 displayed decrease exercise (Fig 3D).


Fig 3. Genome enhancing for endogenous websites.

(A) Schematic of the Cas9 expression constructs. (B) Protein expression degree of Cas9s was measured by western blot. Cells with out Cas9 transfection was used as a detrimental management. (C) Comparability of SaCas9, SmiCas9, Sha2Cas9, and SpeCas9 effectivity for genome enhancing at 13 endogenous loci. Further “g” is added for U6 promoter transcription (n = 3). Underlying information for all abstract statistics may be present in S1 Knowledge. (D) Quantification of enhancing effectivity for SaCas9, SmiCas9, Sha2Cas9, and SpeCas9. Underlying information for all abstract statistics may be present in S1 Knowledge.


Specificity of SmiCas9, Sha2Cas9, and SpeCas9

Subsequent, we in contrast the specificity of SmiCas9, Sha2Cas9, SpeCas9, and SaCas9 utilizing the GFP activation assay. A panel of sgRNAs with dinucleotide mutations alongside the protospacer was generated to detect the specificity of every Cas9. Off-target cleavage is taken into account to have occurred when the mismatched sgRNAs induce GFP expression. Total, SaCas9 and SmiCas9 had negligible off-target results, whereas Sha2Cas9 and SpeCas9 displayed reasonable off-target results (S5 Fig). Particularly, SaCas9 was extremely delicate to mismatches at PAM-proximal and PAM-distal positions however comparatively much less delicate at center positions; SmiCas9 displayed minimal off-target results with mismatches in any respect positions; and Sha2Cas9 and SpeCas9 have been delicate to mismatches at PMA-proximal positions 18 by way of 20 however much less delicate at different positions.

Lately, Tan and colleagues unraveled the crystal construction of the SaCas9/sgRNA–goal DNA advanced and recognized 4 amino acid residues (R245, N413, N419, and R654) forming polar contacts inside a 3.0-Å distance from the goal DNA strand [18]. When a number of of those residues have been changed by alanine, SaCas9 specificity was considerably improved [18]. To analyze whether or not the specificity of Sha2Cas9 may be improved, we used pairwise alignment to establish the corresponding residues (R247, N415, S421, and R656; S6 Fig) and generated single amino acid mutants by alanine substitution. The GFP activation assay revealed that the R247A and N415A mutations might considerably enhance specificity with out compromising the on-target exercise (S7A and S7B Fig). The R656A mutation additionally improved the specificity though this was accompanied by markedly decreased on-target exercise. We launched the R247A and N415A double mutations into Sha2Cas9 to generate a high-fidelity model of Cas9 named Sha2Cas9-HF. The GFP activation assay revealed that double mutations additional improved its specificity (Fig 4A).


Fig 4. Evaluation of Sha2Cas9-HF and SpeCas9-HF specificity.

(A) Schematic of the GFP activation assay for specificity evaluation is proven on the highest. A panel of sgRNAs with dinucleotide mutations is proven beneath. sgRNA actions have been measured primarily based on GFP expression. Mismatches are proven in pink (n = 3). Underlying information for all abstract statistics may be present in S1 Knowledge. (B) Off-targets for EMX1 locus are analyzed by GUIDE-seq. Learn numbers for on- and off-targets are proven on the suitable. Mismatches in contrast with the on-target website are proven and highlighted in coloration.


We concurrently recognized the corresponding residues for SpeCas9 (R247, N415, S421, and R656; S6 Fig) and generated single amino acid mutants by alanine substitution (S8A Fig). The GFP activation assay revealed that the R247A, N415A, and S421A mutations might considerably enhance specificity with out compromising the on-target exercise (S8B Fig). We launched the R247A, N415A, and S421A triple mutations into SpeCas9 to generate a high-fidelity model of Cas9 named SpeCas9-HF. The GFP activation assay revealed that triple mutations additional improved specificity (Fig 4A).

Genome-wide unbiased off-target results of Sha2Cas9, Sha2Cas9-HF, SpeCas9, and SpeCas9-HF have been subsequent evaluated by GUIDE-seq [19]. We evaluated two websites focusing on the EXM1 gene and one website focusing on the RUNX1 gene. 5 days after transfection of the Cas9 plasmid, the sgRNA plasmid, and the GUIDE-seq oligos, we ready libraries for deep sequencing. Sequencing and evaluation confirmed that on-target cleavage occurred for all Cas9 nucleases at 3 targets, as mirrored by the excessive GUIDE-seq learn counts (Fig 4B). Excessive-fidelity variations of Cas9s displayed considerably fewer off-target results than wild-type Cas9s, mirrored by the numbers of off-target websites and off-target learn counts. For instance, SpeCas9 and SpeCas9-HF generated comparable learn counts (225,292 versus 202,764) on the EXM1-sg2 website. SpeCas9 induced 4 off-target websites, whereas SpeCas9-HF induced two off-target websites. For one off-target, SpeCas9 generated 60,331 learn counts, whereas SpeCas9-HF generated 1,061 learn counts. For an additional off-target, SpeCas9 generated 5,634 learn counts, whereas SpeCas9-HF generated 2 learn counts. These information demonstrated that the prevalence of off-target occasions is considerably decrease when utilizing Sha2Cas9-HF and SpeCas9-HF.

Analysis of Sha2Cas9-HF and SpeCas9-HF on-target actions

Subsequent, we in contrast the actions of high-fidelity Cas9s to these of wild-type Cas9s (Sha2Cas9 versus Sha2Cas9-HF; SpeCas9 versus SpeCas9-HF) with a panel of 13 endogenous websites. Western blot evaluation revealed that the protein expression ranges of high-fidelity Cas9s and wild-type Cas9s have been comparable (Fig 3B). All 4 Cas9s generated indels at targets with various efficiencies (Fig 5A). Total, high-fidelity Cas9s and wild-type Cas9s displayed comparable efficiencies (Fig 5B). Nevertheless, completely different efficiencies have been noticed for plenty of targets. For instance, SpeCas9-HF displayed increased effectivity than SpeCas9 on the G5 website, whereas SpeCas9-HF displayed decrease effectivity than SpeCas9 on the G8 website. These information demonstrated that the choice of high-fidelity Cas9s for nucleotides differs from that of wild-type Cas9s for genome enhancing.


Fig 5. Analysis of Sha2Cas9-HF and SpeCas9-HF on-target actions.

(A) Comparability of actions of high-fidelity Cas9s to the wild-type Cas9s (n = 3). The goal sequences are proven on the left. PAM is underlined. If the primary nucleotide is C or T, extra “g” is added for U6 promoter transcription. Underlying information for all abstract statistics may be present in S1 Knowledge. (B) Quantification of enhancing effectivity for SaCas9, SmiCas9, Sha2Cas9, and SpeCas9. Underlying information for all abstract statistics may be present in S1 Knowledge.



Totally different nucleotide preferences have been noticed amongst pure Cas9 nucleases. For instance, SpCas9 favors G-rich sequences however disfavors T-rich sequences [8]; AsCas12a favors A-rich sequences however disfavors G-rich sequences [9]. One doable technique to realize excessive effectivity of genome enhancing is to harness a number of pure Cas nucleases for genome enhancing, and a group of those nucleases might cowl all doable sequences. Quite a few Cas nucleases, akin to SaCas9 [17], NmeCas9 [20], CjCas9 [21], AaCas12b [22], and Cas12f1 [23,24], have been harnessed for genome enhancing. We beforehand developed BlatCas9 [25], SauriCas9 [13], SlugCas9 [14], and SchCas9 [15] for genome enhancing. On this examine, we additional expanded the Cas repertoire by creating SmiCas9, Sha2Cas9, and SpeCas9. Importantly, they include a compact genome, facilitating supply by a single adeno-associated virus (AAV) for in vivo genome enhancing. These newly developed Cas9s will improve our capacity to realize excessive effectivity genome enhancing.

Totally different nucleotide preferences have additionally been noticed between pure Cas9s and their engineered variants. We and others beforehand screened hundreds of sgRNA actions for SpCas9 and its engineered variants and noticed completely different nucleotide preferences [8,26]. For instance, SpCas9 barely prefers A and G at sgRNA place 10, whereas SpCas9-HF1 strongly prefers C at this place [8]. On this examine, we generated two high-fidelity variations of Cas9s. Though they solely include 2 or 3 amino acid modifications, distinct nucleotide preferences have been noticed for plenty of targets. Due to this fact, engineered Cas9s not solely change specificity or focusing on scope [2730] but in addition change nucleotide preferences.

Cas9s with versatile PAMs are essential for precision positioning. Along with SpCas9, a number of different pure Cas nucleases with dinucleotide PAMs have been recognized, together with FnCas9 [31], Nme2Cas9 [32], SauriCas9 [13], SlugCas9 [14], SchCas9 [15], and AaCas12b [22]. On this examine, we recognized the serine residue similar to SaCas9 N986 related to the easy NNGG PAM requirement. This PAM happens, on common, as soon as in each roughly 8 randomly chosen genomic loci. We additional recognized three amino acids that decided the NNGG PAM requirement of SaCas9. With the continual enlargement of the Cas9 database, our technique will supply a clue to establish extra SaCas9 orthologs with NNGG PAMs.

Supplies and strategies

Plasmid development

Cas9 expression plasmid development: The plasmid pX601 (Addgene#61591) was amplified by the primers px601-F/px601-R to acquire the pX601 spine. The human codon–optimized Cas9 gene (S1 Desk) was synthesized by HuaGene (Shanghai, China) and cloned into the pX601 spine by the NEBuilder meeting software (NEB) in response to the producer’s directions. Sequences of every Cas9 have been confirmed by Sanger sequencing (GENEWIZ, Suzhou, China).

sgRNA expression plasmid development: sgRNA expression plasmids have been constructed by ligating sgRNA into the Bsa1-digested hU6-Sa_tracr plasmid. The primer sequences and goal sequences are listed in S2 and S3 Tables, respectively.


GUIDE-seq experiments have been carried out as described beforehand [19], with minor modifications. Briefly, 2 × 105 HEK293T cells have been transfected with 500 ng of SchCas9/Sa-SchCas9, 500 ng of sgRNA plasmids, and 100 pmol of annealed GUIDE-seq oligonucleotides by electroporation after which seeded into 6 wells. The electroporation voltage, width, and the variety of pulses have been 1,150 V, 30 ms, and 1 pulse, respectively. Genomic DNA was extracted with the DNeasy Blood and Tissue package (QIAGEN) 6 days after transfection in response to the producer’s protocol. The genome library was ready and subjected to deep sequencing [19].

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