US20190218532A1 - Streptococcus Canis Cas9 as a Genome Engineering Platform with Novel PAM Specificity - Google Patents

Streptococcus Canis Cas9 as a Genome Engineering Platform with Novel PAM Specificity Download PDF

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US20190218532A1
US20190218532A1 US16/136,238 US201816136238A US2019218532A1 US 20190218532 A1 US20190218532 A1 US 20190218532A1 US 201816136238 A US201816136238 A US 201816136238A US 2019218532 A1 US2019218532 A1 US 2019218532A1
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pam
cas9
sccas9
crispr
streptococcus
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Joseph M. Jacobson
Noah Michael Jakimo
Pranam Chatterjee
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Massachusetts Institute of Technology
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Priority to US16/689,071 priority patent/US11453865B2/en
Priority to US17/683,299 priority patent/US12054755B2/en
Priority to US17/841,639 priority patent/US20230193229A1/en
Priority to US17/855,507 priority patent/US11697808B2/en
Priority to US18/220,808 priority patent/US20240141309A1/en
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Definitions

  • the present invention relates to genome editing and, in particular, to a Streptococcus Cas9 ortholog having novel PAM specificity, along with variants and uses thereof.
  • RNA-guided DNA endonucleases of the CRISPR-Cas system, such as Cas9[M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science 337, 816-821 (2012)] and Cpf1 (also known as Cas12a) [B. Zetsche, J. S. Gootenberg, O. O. Abudayyeh, I. M. Slaymaker, K. S.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the invention includes a novel Streptococcus Cas9 ortholog and its engineered variants, possessing novel PAM specificity.
  • the invention includes a novel DNA-interacting loop domain within Streptococcus canis Cas9 (ScCas9), and other Cas9 orthologs.
  • the invention includes a method of altering expression of at least one gene product by employing Streptococcus canis Cas9 (ScCas9) and other Cas9 orthologs.
  • the invention is an isolated Streptococcus canis Cas9 (ScCas9) protein or transgene expression thereof.
  • the protein may include at least one of the mutations K857A, K1012A, R1069A, N507A, R671A, Q705A, Q935A, N702A, M704A, Q705A, and H708A.
  • the invention is CRISPR-associated DNA endonuclease with PAM interacting domain (PID) amino acid sequences that are at least 80% identical to that of the isolated Streptococcus canis Cas9 (ScCas9) protein.
  • PAM interacting domain PAM interacting domain
  • the endonuclease may have a PAM specificity of “NNGT” or “NNNGT”, may comprise a 10 amino acid loop insertion of “IKHRKRTTKL” [SEQ ID No. 4], or may comprise a 2 amino acid insertion of “KQ” two positions upstream of the first critical arginine (R) residue for PAM binding.
  • the invention is an isolated, engineered Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophdus Cas9, or Cpf1 protein with a PID as either the PID amino acid composition of the isolated Streptococcus canis Cas9 (ScCas9) protein or of CRISPR-associated DNA endonucleases with PAM interacting domain (PID) amino acid sequences that are at least 80% identical to that of the isolated Streptococcus canis Cas9 (ScCas9) protein.
  • the protein may include at least one of the amino acid insertions “IKHRKRTTKL”_[SEQ ID No.
  • the invention is a DNA-interacting loop domain within ScCas9, or a Cas9 ortholog, that facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.
  • the Cas9 orthologs may comprise Streptococcus gordonii or Streptococcus angionosis.
  • the invention is a method for altering expression of at least one gene product by employing Streptococcus canis Cas9 (ScCas9) endonucleases in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence.
  • SpCas9 Streptococcus canis Cas9
  • the invention is a method of altering expression of at least one gene product comprising: introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product, an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising (a) a regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR system guide RNA that hybridizes with the target sequence, and (b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding one or more of the proteins in claims 1 - 10 , wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and one or more of the proteins in claims 1 - 10 cleave the DNA molecule, whereby expression of the at least one gene product is altered; and,
  • FIG. 1 depicts the global pairwise sequence alignment of Streptococcus pyogenes Cas9 (SpCas9) and Streptococcus canis Cas9 (ScCas9).
  • FIG. 2 illustrates the DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, demonstrating that this loop forms expected sequence unspecific contacts with the negatively-charged phosphate backbone of the target DNA strand.
  • FIG. 3 depicts a WebLogo for sequences found at the 3′ end of protospacer targets identified in plasmid and viral genomes using Type II spacer sequences within Streptococcus canis as BLAST queries.
  • FIG. 4 illustrates PAM determination of engineered ScCas9 variants by showing PAM binding enrichment on a 5′-NNNNNN-3′ (8N) PAM library.
  • FIG. 5 is a graph illustrating an examination of PAM preference for ScCas9.
  • FIGS. 6-8 demonstrate ScCas PAM specificity in human cells, wherein:
  • FIGS. 9-12 demonstrate ScCas9 performance as a genome editing tool, wherein:
  • FIGS. 13 and 14 depict the relationship of ScCas9 to other Streptococcus orthologs, wherein:
  • FIG. 15 depicts SPAMALOT PAM Predictions for Streptococcus Cas9 Orthologs.
  • FIG. 16 is a schematic depicting an example workflow to knockout a gene in cell culture, using ScCas9 according to an aspect of the invention.
  • the invention is an addition to the family of CRISPR-Cas9 systems repurposed for genome engineering and regulation applications.
  • the invention comprises the usage of Streptococcus canis Cas9 (ScCas9) endonuclease in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence, promoting new flexibility in target selection.
  • ScCas9 Streptococcus canis Cas9
  • the invention is a novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii (Uniprot A0A134D9V8) and Streptococcus angionosis (Uniprot F5U0T2), that may facilitate a divergent PAM sequence from the canonical “NGG” PAM of SpCas9.
  • ScCas9 (UniProt I7QXF2) possesses 89.2% sequence similarity to Sp-Cas9. Despite such homology, ScCas9 prefers a more minimal 5′-NNG-3′ PAM. To explain this divergence, two significant insertions were identified within its open reading frame (ORF) that differentiate ScCas9 from SpCas9 and contribute to its PAM-recognition flexibility. ScCas9 can efficiently and accurately edit genomic DNA in mammalian cells.
  • ORF open reading frame
  • a bioinformatics workflow to identify the PAM specificity of ScCas9 in silico involves the alignment of the spacer sequences within the CRISPR cassette of Streptococcus canis with potential protospacers found within the phage and/or other genome databases. As the PAM lies immediately adjacent to the protospacer sequence, these sequences can be conglomerated and weighted based on the number of mismatches to infer bases that are overrepresented at each position [Ran, F. A. et al., “In vivo genome editing using Staphylococcus aureus Cas9”, Nature 520, 186-191 (2015); Crooks, G. E. et al. “WebLogo: a sequence logo generator”, Genome Res. 14, 1188-1190 (2004)].
  • FIG. 1 depicts the global pairwise amino acid sequence alignment of Streptococcus pyogenes Cas9 (SpCas9) (Uniprot Q99ZW2) and ScCas9 (Uniprot I7QXF2).
  • ScCas9 contains two notable insertions, one positive-charged insertion 110 in the REC domain (367-376) and another KQ insertion 120 in the PAM-interacting domain (1337-1338), as indicated.
  • the 10-residue loop not found in SpCas9, despite otherwise remarkable homology, consists of 8 positively charged amino acids (KHRKRTTK) flanked by two neutral amino acids (I and L).
  • the novel REC motif is inserted into PDB 4OO8.
  • the 367-376 insertion demonstrates a loop-like structure 210.
  • Several of its positive-charged residues 220 come in close proximity to the target DNA near the PAM 230.
  • the novel loop domain can be inserted into the open reading frame (ORF) of SpCas9, and all characterized Cas9 orthologs, such as Streptococcus thermophilus (Uniprot G3ECR1), and other CRISPR endonucleases, such as Cpf1 (Uniprot U2UMQ6 and A0Q7Q2), for the generation of altered PAM specificities through increased protein-DNA interactions.
  • ORF open reading frame
  • FIG. 3 is a WebLogo for sequences found at the 3′ end of protospacer targets identified in plasmid and viral genomes using Type II spacer sequences within Streptococcus canis as BLAST queries.
  • PAM binding sequence of ScCas9 was validated utilizing an existent positive selection bacterial screen based on GFP expression conditioned on PAM binding, termed PAM-SCALAR [R. T. Leenay, K. R. Maksimchuk, R. A. Slotkowski, R. N. Agrawal, A. A. Gomaa, et al., “Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems”, Mol. Cell 62, 137-147 (2016)].
  • a plasmid library containing the target sequence followed by a randomized 5′-NNNNNN-3′ (8N) PAM sequence was bound by a nuclease-deficient ScCas9 (and dSpCas9 as a control) and an sgRNA both specific to the target sequence and general for SpCas9 and ScCas9, allowing for the repression of lacI and expression of GFP.
  • Plasmid DNA from FACS-sorted GFP-positive cells and pre-sorted cells were extracted and amplified, and enriched PAM sequences were identified by Sanger sequencing, and visualized utilizing DNA chromatograms. The results provided initial evidence that ScCas9 can bind to the minimal 5′-NNG-3′ PAM, distinct to that of SpCas9's 5′-NGG-3′.
  • FIGS. 4 and 5 depict aspects of PAM determination of engineered ScCas9 variants.
  • FIG. 4 illustrates PAM binding enrichment on a 5′-NNNNNN-3′ (8N) PAM library.
  • PAM profiles are represented by Sanger sequencing chromatograms via amplification of PAM region following plasmid extraction of GFP+ E. coli cells.
  • the previously described insertions may contribute to the flexibility permitting ScCas9 to bind to the minimal 5′-NNG-3′ PAM, distinct to that of SpCas9's 5′-NGG-3′.
  • ScCas9 was engineered to remove either insertion or both, and subjected these variants to the same screen.
  • FIG. 5 is a graph illustrating an examination of PAM preference for ScCas9.
  • PAMs For individual PAMs, all four bases were iterated at a single position (2, 4, and 5). Each PAM-containing plasmid was electroporated in duplicates, subjected to FACS analysis, and gated for GFP expression. Subsequently, GFP expression levels were averaged. Standard deviation was used to calculate error bars and statistical significance analysis was conducted using a two-tailed Student's t-test as compared to the negative control.
  • KQ amino acid insertion
  • R arginine residues critical for PAM binding of Cas9. It is likely that this insertion shifts the length and alters the specificity of the PAM adjacent to the target sequence.
  • a preferred embodiment of this invention enables both the insertion of the KQ motif one amino acid upstream of the first critical arginine residue in SpCas9 to alter its PAM specificity, as well as the removal of the KQ motif in ScCas9 for a similar purpose.
  • these residue-specific mutations that decrease off-target activity while maintaining robust on-target nuclease activity can be applied to the ORF of ScCas9 to generate a hyper-accurate ScCas9 endonuclease.
  • the invention is compatible with existing delivery methods used for other CRISPR-Cas9 systems including, but not limited to, electroporation, lipofection, viral infection, and nanoparticle injection.
  • Embodiments can co-deliver the invention as a coding nucleic acid or protein, along with a gRNA. Components can also be stably expressed in cells.
  • the PAM specificity of ScCas9 was compared to SpCas9 in human cells by co-transfecting HEK293T cells with plasmids expressing these variants along with sgRNAs directed to a native genomic locus (VEGFA) with varying PAM sequences (Table S 1 ). Editing efficiency was first tested at a site containing an overlapping PAM (5′-GGGT-3′). After 48 hours post-transfection, gene modification rates, as detected bythe T7E1 assay, demonstrated comparable editing activities of SpCas9, ScCas9, and ScCas9 ⁇ Loop ⁇ KQ. Additionally sgRNAs to sites with various non-overlapping 5′-NNGN-3′ PAM sequences were constructed.
  • FIG. 6 depicts a T7E1 analysis of indels produced at VEGFA loci with indicated PAM sequences.
  • the Cas9 used is indicated above each lane. All samples were performed in biological duplicates.
  • SpCas9, ScCas9, and ScCas9 ⁇ Loop ⁇ KQ were transfected without targeting guide RNA vectors.
  • FIG. 7 is a graph depicting an example quantitative analysis of T7E1 products. Unprocessed gel images were quantified by line scan analysis using Fiji [J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, et al., “Fiji: an open-source platform for biological-image analysis”, Nat.
  • ScCas9 ⁇ Loop ⁇ KQ was able to cleave at the 5′-NGG-3′ target, along with significant activity on the 5′-NNGA-3′ target, with reduced gene modification levels at all other 5′-NNGN-3′ targets ( FIGS. 6 and 7 ).
  • ScCas9 can serve as an effective alternative to SpCas9 for genome editing in mammalian cells, both at overlapping 5′-NGG-3′ and more minimal 5′-NNGN-3′ PAM sequences.
  • the PAM specificity of ScCas9 base editors was assessed by using a synthetic Traffic Light Reporter (TLR) [M. T. Certo, B. Y. Ryu, J. E. Annis, M. Garibov, J. Jarjour, et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nat. Methods 8, 671-676 (2011)] plasmid, containing an early stop codon upstream of a GFP ORF and downstream of an mCherry ORF.
  • GFP+ cells were calculated as a percentage of mCherry+ cells for indicated PAM sequences using the Traffic Light Reporter [M. T. Certo, B. Y. Ryu, J. E. Annis, M. Garibov, J. Jarjour, et al., “Tracking genome engineering outcome at individual DNA breakpoints”, Nat. Methods 8, 671-676 (2011)] with an early stop codon. All samples were performed in duplicates and quantified percentages were averaged. Standard deviation was used to calculate error bars and statistical significance analysis was conducted using a two-tailed Student's t-test.
  • Each of these three sites additionally possesses a single off-target that has been particularly difficult to mediate via engineering of high-fidelity Cas9 variants [I. M. Slaymaker, L. Gao, B. Zetsche, D. A. Scott, W. X. Yan, et al., “Rationally engineered Cas9 Nucleases with improved specificity”, Science 351, 84-88 (2016); B. P. Kleinstiver, V. Pattanayak, M. S. Prew, S. Q. Tsai, N. T. Nguyen, et al., “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects”, Nature 529, 490-495 (2016); J. S.
  • ScCas9's activity was analyzed on these off-targets. After co-transfection of sgRNAs to the three aforementioned sites alongside both SpCas9 and ScCas9, genomic DNA flanking both the on-target and difficult off-target sequences was amplified to assess their genome modification activities.
  • FIG. 9 is a graph of results from quantitative analysis of T7E1 products for indicated genomic on- and off-target editing. All samples were performed in duplicates and quantified modification values were averaged. Standard deviation was used to calculate error bars and statistical significance analysis was conducted using a two-tailed Student's t-test as compared to each negative control. Mismatched positions 910, 920, 930, 940, 950 within the spacer sequence are highlighted.
  • FIG. 10 is an efficiency heatmap of the mismatch tolerance assay. Quantified modification efficiencies, as assessed by the T7E1 assay, are exhibited for each labeled single or double mismatch in the sgRNA sequence for each indicated PAM. Across all of the four PAM targets, ScCas9 does tolerate mismatches within the middle of the crRNA sequence, with highest efficiencies reported for the 5′-NTG-3′ target. SpCas9 expectedly demonstrates negligible genome modification activity on the 5′-NCG-3′ and 5′-NTG-3′ targets, but weakly tolerates single and double mismatches across the entire crRNA sequence, with reduced tolerance in the seed region, for the standard 5′-NGG-3′ target, corroborating previous mismatch tolerance studies [J. S.
  • ScCas9 exhibits a similar mismatch tolerance profile to SpCas9 on the 5′-NAG-3′ target, albeit with a higher reported on-target efficiency.
  • ScCas9 Genome Editing Capabilities were evaluated for the ability to modify a variety of gene targets for a handful of different PAM sequences was evaluated. sgRNAs to 24 targets within 9 endogenous genes in HEK293T cells were constructed, and on-target gene modification was evaluated utilizing the T7E1 assay. The results demonstrate that ScCas9 maintains comparable efficiencies to that of SpCas9 on 5′-NGG-3′ sequences, as well as on selected 5′-NNG-3′ PAM targets, supporting the previous findings ( FIG. 7 ).
  • FIG. 11 is a dot plot of on-target modification percentages at various gene targets for indicated PAM as assessed by the T7E1 assay. Duplicate modification percentages were averaged.
  • SpCas9 expectedly performs efficiently on 5′-NGG-3′ and weakly on 5′-NAG-3′ tar-gets, but demonstrates negligible editing capabilities on 5′-NCG-3′ and 5′-NTG-3′ PAM sequences, as previously demonstrated.
  • ScCas9 performed less effectively on selected target sequences in the Hemoglobin subunit delta (HBD) gene, while demonstrating higher efficiencies on 5′-NNG-3′ sequences in VEGFA and DNMT1, for example.
  • HBD Hemoglobin subunit delta
  • BEEP Base Editing Evaluation Program
  • ScCas9 base editors perform efficiently on the non-5′-NGG-3′ targets, as compared to SpCas9 ( FIGS. 8 and 12 ), ScCas9 is less effective at editing 5′-NGG-3′ genomic targets than SpCas9 for both architectures, indicating that further development is necessary for broad usage of ScCas9 base editors.
  • FIG. 13 depicts PAM binding enrichment on a 5′-NNNNNNNN-3′ PAM library of ScCas9-like SpCas9 variants.
  • the PAM-SCANR screen (23) was applied to variants of SpCas9 containing either the loop or KQ insertions, or both.
  • FIG. 14 illustrates FACS analysis of binding at an 5′-NGG-3′ PAM. All samples were performed in duplicates and averaged. Standard deviation was used to calculate error bars.
  • S. canis has been reported to infect dogs, cats, cows, and humans, and has been im-plicated as an adjacent evolutionary neighbor of S. pyogenes, as evidenced by various phylogenetic analyses [T. Lef'ebure, V. P. Richards, P. Lang, P. Pavinski-Bitar, M. J. Stanhope, “Gene Repertoire Evolution of Streptococcus pyogenes Inferred from Phylogenomic Analysis with Streptococcus canis and Streptococcus dysgalactiae”, PLOS ONE 7, e37607 (2012); 32. V. P. Richards, R. N. Zadoks, P. D. Pavinski Bitar, T.
  • ScCas9 PID is mostly composed of disjoint sequences from other orthologs, such as those from S. phocae, S. varani, and S. equinis. Additional LGT events between these orthologs, as opposed to isolated divergence, more likely explain the differences between ScCas9 and SpCas9.
  • FIG. 15 depicts SPAMALOT PAM Predictions for Streptococcus Cas9 Orthologs.
  • Spacer sequences found within the Type II CRISPR cassettes associated with Cas9 ORFs from specified Streptococcus genomes were aligned to Streptococcus phage genomes to generate spacer-protospacer mappings.
  • WebLogos, labeled with the relevant species, genome, and CRISPR repeat, were generated for sequences found at the 3′ end of candidate protospacer targets with no more than two mismatches (2 mm).
  • Shown in FIG. 15 are PAM predictions for experimentally validated Cas9 PAM sequences 1510 in previous studies, novel PAM predictions of alternate S. thermophilus Cas9 orthologs 1520 with putative divergent specificities, and novel PAM predictions of uncharacterized Streptococcus orthologs 1530 with distinct specificities.
  • FIG. 15 1510 shows that resulting WebLogos accurately reflect the known PAM specificities of Cas9 from S. canis (this work), S. pyogenes, S. thermophilus, and S. mutans [ S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, J. A. Doudna, “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9”, Nature 507, 62-67 (2014); M. Muller, C. M. Lee, G. Gasiunas, T. H. Davis, T. J.
  • thermophilus WebLogo upon subtle specificity changes that traverse intermediate WebLogos among them.
  • S. oralis WebLogos that also share this repeat, as well as unique putative PAM specificities associated with CRISPR cassettes containing S. mutans -like repeats from the S. oralis, S. equinis, and S. pseudopneumoniae genomes ( FIG. 15 1530 ).
  • PACE phage assisted continuous evolution
  • SPAMALOT is established as an accessible resource that is shared with the community for application to CRISPR cassettes from other genera. Future development will include broadening the scope of candidate targets beyond genus-associated phage to capture additional se-quences that could be beneficial targets, such as lysogens in species that host the same phage. It is hoped that this pipeline can be utilized to more efficiently validate and engineer PAM specificities that expand the targeting range of CRISPR, especially for strictly PAM-constrained technologies such as base editing and homology repair induction.
  • ScCas9 does not require any alterations to the sgRNA of SpCas9, and due to its significant sequence homology with SpCas9, identical modifications from previous studies [I. M. Slaymaker, L. Gao, B. Zetsche, D. A. Scott, W. X. Yan, et al., “Rationally engineered Cas9 Nucleases with improved specificity”, Science 351, 84-88 (2016); B. P. Kleinstiver, V. Pattanayak, M. S. Prew, S. Q. Tsai, N. T.
  • the Cas9 from Streptococcus canis was codon optimized for E. Coli, ordered as multiple gBlocks from Integrated DNA Technologies (IDT), and assembled using Golden Gate Assembly.
  • the pSF-EF1-Alpha-Cas9WT-EMCV-Puro (OG3569) plasmid for human expression of SpCas9 was purchased from Oxford Genetics, and the ORFs of Cas9 variants were individually amplified by PCR to generate 35 bp extensions for subsequent Gibson Assembly into the OG3569 backbone.
  • Engineering of the coding sequence of ScCas9 and SpCas9 for removal or insertion of motifs was conducted using either the Q5 Site-Directed Mutagenesis Kit (NEB) or Gibson Assembly.
  • sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Table S1) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately down-stream of the human U6 promoter sequence. Assembled constructs were transformed into 50 , ⁇ L NEB Turbo Competent E. coli cells, and plated onto LB agar supplemented with the appropriate antibiotic for subsequent sequence verification of colonies and plasmid purification.
  • NEB KLD enzyme mix
  • Nuclease-deficient mutations (D10A and H850A) were introduced to the ScCas9 variants using Gibson Assembly as previously described.
  • the provided BW25113 cells were made electrocompetent using standard glycerol wash and resuspension protocols.
  • the outgrowth was diluted 1:100, grown to ABS600 of 0.6 in Kan+Crb LB liquid media, and made electrocompetent.
  • Indicated dCas9 plasmids with resistance to chloramphenicol (Chl), were electroporated in duplicates into the electrocompetent cells harboring both the PAM library and sgRNA plasmids, outgrown, and collected in 5 mL Kan+Crb+Chl LB media. Overnight cultures were diluted to an ABS600 of 0.01 and cultured to an OD600 of 0.2. Cultures were analyzed and sorted on a FACSAria machine (Becton Dickinson). Events were gated based on forward scatter and side scatter and fluorescence was measured in the FITC channel (488 nm laser for excitation, 530/30 filter for detection), with at least 30,000 gated events for data analysis.
  • FITC channel 488 nm laser for excitation, 530/30 filter for detection
  • Sorted GFP-positive cells were grown to sufficient density, and plasmids from the pre-sorted and sorted populations were then isolated, and the region flanking the nucleotide library was PCR amplified and submitted for Sanger sequencing (Genewiz). Bacteria harboring non-library PAM plasmids, performed in duplicates, were analyzed by FACS following electroporation and overnight incubation, and represented as the percent of GFP-positive cells in the population, utilizing standard deviation to calculate error bars. Additional details on the PAM-SCALAR assay can be found in Leenay, et al. [R. T. Leenay, K. R. Maksimchuk, R. A. Slotkowski, R. N. Agrawal, A. A. Gomaa, et al., “Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems”, Mol. Cell 62, 137-147 (2016].
  • FIG. 16 is a schematic depicting an example workflow to knockout a gene in cell culture, using ScCas9 according to an aspect of the invention.
  • an example workflow to knockout a gene in cell culture begins with the user's preferred method of selecting a gRNA target adjacent to an ScCas9-specified PAM around a gene of interest from a FASTA sequence file corresponding to this region.
  • a bicistronic vector containing both the gRNA under the control of a U6 promoter and either the coding sequence of the invention or that of its engineered variants, under the control of a mammalian constitutive promoter is constructed using existing assembly and cloning techniques.
  • the plasmid can be delivered using a standard lipofection reagent (e.g. TransIT-X2 from Mirus Bio LLC) into cell culture. After roughly two days of incubation, individual cells are harvested for genomic extraction to allow an approximately one kilobase (kb) window around the target to be amplified via polymerase chain reaction (PCR). The PCR product is ligated into a bacterial plasmid with a drug selection marker through blunt end cloning and transformed into E. coli. Bacterial colonies are subsequently picked for monoclonal Sanger sequencing and can be carried out by services such as Genewiz.
  • a standard lipofection reagent e.g. TransIT-X2 from Mirus Bio LLC
  • PCR polymerase chain reaction
  • HEK293T cells were maintained in DMEM supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • sgRNA plasmid (500 ng) and effector (nuclease, BE3, or ABE(7.10)) plasmid (500 ng) were transfected into cells as duplicates (2 ⁇ 105/well in a 24-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). After 48 hours post-transfection, genomic DNA was extracted using QuickExtract Solution (Epicentre), and genomic loci were amplified by PCR utilizing the KAPA HiFi HotStart ReadyMix (Kapa Biosystems).
  • amplicons were purified and submitted for Sanger sequencing (Genewiz).
  • the T7E1 reaction was conducted according to the manufacturer's instructions and equal volumes of products were analyzed on a 2% agarose gel stained with SYBR Safe (Thermo Fisher Scientific). Unprocessed gel image files were analyzed in Fiji [J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, et al., “Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682 (2012)].
  • HEK293T cells were maintained as previously described, and transfected with the corresponding sgRNA plasmids (333 ng), ABE7.10 plasmids (333 ng), and synthetically constructed TLR plasmids (333 ng) into cells as duplicates (2 ⁇ 105/well in a 24-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco).
  • TLR spacer sequence is 5′-TTCTGTAGTCGACGGTACCG-3′ [SEQ ID No. 6].
  • the Base Editing Evaluation Program (BEEP) was written in Python, employing the pandas data manipulation library and BioPython package. As inputs, the program requires a sample ab 1 file, a negative control ab1 file, a target sequence, as well as the position of the specified base conversion, either handled as a .csv file for multiple sample analysis or for individual samples on the command line. Briefly, the provided target sequences are aligned to the base-calls of each input ab1 file to determine the absolute position of the target within the file. Subsequently, the peak values for each base at the indicated position in the spacer are obtained, and the editing percentage of the specified base conversion is calculated. Finally, a separate function normalizes the editing percentage to that of the negative control ab 1 file to account for background signals of each base. The final base conversion percentage is outputted to the same .csv file for downstream analysis.
  • the isolated Streptococcus canis Cas9 (ScCas9) protein comprising one or more of the following mutations: K857A, K1012A, R1069A, N507A, R671A, Q705A, Q935A, N702A, M704A, Q705A, H708A.
  • SpCas9 protein with its PID as either the PID amino acid composition of the isolated Streptococcus canis Cas9 (ScCas9) protein or of CRISPR-associated DNA endonucleases with PAM interacting domain (PID) amino acid sequences that are at least 80% identical to that of the isolated Streptococcus canis Cas9 (ScCas9) protein.
  • PID PAM interacting domain
  • a method of altering expression of at least one gene product comprising: introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product, an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising one or more vectors comprising (a) a regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR system guide RNA that hybridizes with the target sequence, and (b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding one or more of the proteins in (1)-(10), wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and one or more of the proteins in (1)-(10) clea
  • the present invention demonstrates the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. Affinity to minimal 5′ -NNG-3′ PAM sequences and the accurate editing capabilities of the ortholog in both bacterial and human cells have been demonstrated.
  • an automated bioinformatics pipeline the Search for PAMs by ALignment Of Targets (SPAMALOT) further explores the microbial PAM diversity of otherwise-overlooked Streptococcus Cas9 orthologs. The results establish that ScCas9 can be utilized both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.

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US17/841,639 US20230193229A1 (en) 2017-09-19 2022-06-15 Applications of Recombined ScCas9 Enzymes for PAM-free DNA Modification
US17/855,507 US11697808B2 (en) 2017-09-19 2022-06-30 Applications of engineered Streptococcus canis Cas9 variants on single-base PAM targets
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