Patent Application
20250179457
This invention pertains to Lachnospiraceae bacterium Cas12a based CRISPR genes, polypeptides encoded by the same, mammalian cell lines that stably express Cas 12, crRNAs and the use of these materials in compositions of CRISPR-Cas12a systems and methods.
This application was filed with a Sequence Listing XML in ST.26 XML format in accordance with 37 C.F.R. § 1.831. The Sequence Listing XML file submitted in the USPTO Patent Center, “013670-9059-US03_sequence_listing_xml_17-FEB-2025.xml,” was created on Feb. 17, 2025, contains 73 sequences, has a file size of 116 kilobytes (118,784 bytes), and is incorporated by reference in its entirety into the specification.
Cas12a (previously named Cpf1) is a class 2/type V CRISPR RNA-guided endonuclease. Zetsche et al., Cell 163: 1-13 (2015). Cas12a is an effective nuclease used for genome editing and is an alternative to the Cas9 enzyme. Cas12a is a ˜1300 amino acid protein and is slightly smaller than Cas9 from S. pyogenes. The Cas12 system does not utilize a separate tracrRNA, and only requires a single short crRNA of 40-45 nucleotides in length that both specifies target DNA sequence and directs binding of the RNA to the Cas12a nuclease. Hur et al., Nature Biotechnology 34: 807-808 (2016). The PAM recognition sequence of Cas12a is TTTV which allows for expanded coverage in adenine and thymidine rich areas of the genome that Cas9 cannot access.
Cleavage by Cas12a results in a staggered double-stranded break in the DNA with 4-5 nucleotide overhangs, which leaves staggered ends distal to the PAM site. Gao et al., Cell Research 26: 901-913 (2016). These double stranded breaks can then be repaired via non-homologous end joining (NHEJ) which often leads to mutations or insertions/deletions at the cut site or site or homology directed repair (HDR) which can generate precise editing events. Furthermore, when Cas12a cleaves, it does so further away from PAM than Cas9, which is also further away from the target site. As a result, the protospacer, and especially the seed sequence of the protospacer, are less likely to be edited, thereby leaving open the potential for a second round of cleavage if the desired repair event doesn't occur the first time.
LbCas12a is an RNA guided endonuclease from the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) adaptive immune system from Lachnospiraceae bacterium ND2006 (Lb) species. Cas12a nucleases are classified as a class 2 type V CRISPR system that provide a staggered DNA double-stranded break with a 5-nucleotide 5′-overhang when complexed with a CRISPR RNA (crRNA) [1]. The LbCas12a:crRNA complex is referred to as a CRISPR ribonucleoprotein (RNP) complex.
LbCas12a, along with AsCas12a (Acidaminococcus sp. BV3L6), was first characterized in 2015 [1] and since have successfully been used for genome editing in eukaryotic cells [1-8]. The two Cas12a variants, As and Lb, share a 34% sequence identity and have both been crystallized by the Nureki group [9-10]. The RuvC and Nuc domains of both variants of Cas12a are structurally similar and cleave the target DNA by similar mechanisms [9-10]. Both variants recognize the TTTV as the canonical PAM and have been shown to tolerate CTTV, TCTV and TTCV as non-canonical PAMs [6, 10].
Engineered Cas12a proteins have been reported by Zhang and coworkers that show altered PAM specificities [11]. Their primary objective was to perform a structure guided mutagenesis screen on AsCas12a, followed by mirror mutations in LbCas12a. This resulted in two mutant variants, AsCas12a-S542R/K607R and AsCas12a-S542R/K548V/N552R, which recognized the PAMs TYCV and TATV, respectively. These mutants retained the high specificity of these CRISPR proteins and introducing these mutations into LbCas12a (G532R/K595R and G532R/K538V/T542R, respectively) resulted in similar PAM-altering specificity [11].
This invention pertains to Cas12a CRISPR genes and mutants, polypeptides encoded by the same, mammalian cell lines that stably express Cas12a and their use in compositions of CIRSPR-Cas12a systems and methods. Examples are shown employing the Cpf1 systems from Lachnospiraceae bacterium ND2006 (Lb) however this is not intended to limit scope, which extends to Cas12a homologs or orthologs isolated from other species.
Additionally, the present invention pertains to the ability to cleave double-stranded DNA of living organisms at precise locations with the CRISPR/LbCas12a nuclease system. Additionally, the present invention describes the single amino acid substitution of LbCas12a that enhances genome editing efficiency as compared to wild type Cas12a variants, LbCas12a and AsCas12a, and is claimed as LbCas12a-E795L. This invention also includes six other mutants of LbCas12a, N527R, D559P, N527R/D559P, N527R/E795L, D559P/E795L and N527R/D559P/E795L, that showed similar genome editing as wild type LbCas12a.
In a first embodiment an isolated mutant Cas12a protein is provided. The isolated mutant Cas12a protein is active in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein endonuclease system (“CRISPR/Cas12a endonuclease system”). The CRISPR/Cas12a endonuclease system maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system. In another aspect the Cas12a protein is isolated from Lachnospiraceae bacterium ND2006 (Lb).
In a second embodiment, an isolated ribonucleoprotein (RNP) complex is provided. The RNP complex includes a mutant Cas12a protein and a crRNA. The isolated ribonucleoprotein complex is active as a CRISPR/Cas12a endonuclease system, wherein the resultant CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system.
In a third embodiment, an isolated nucleic acid encoding a mutant Cas12a protein is provided. The mutant Cas12a protein is active in a CRISPR/Cas12a endonuclease system, wherein the CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to wild-type CRISPR/Cas12a endonuclease system.
In a fourth embodiment, a CRISPR/Cas12a endonuclease system is provided. The CRISPR/Cas12a endonuclease system includes a mutant Cas12a protein and a crRNA. The CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to wild-type CRISPR/Cas12a endonuclease system.
In a fifth embodiment, a method of performing gene editing having maintained on-target editing activity is provided. The method includes the step of contacting a candidate editing DNA target site locus with an active CRISPR/Cas12a endonuclease system having a mutant Cas12 a protein complexed with an appropriate crRNA. Said interaction can occur an any context, for example, in a live animal, in live cells, or an isolated DNA in vitro.
In another embodiment the CRISPR/Cas12a endonuclease system of the present invention displays maintained on-target editing activity relative to a wild type CRISPR/Cas endonuclease system and may display reduced off-target editing activity when compared to wild type CRISPR/Cas endonuclease systems. In another aspect the CRISPR/Cas12a endonuclease system of the present invention displays maintained on-target editing activity relative to a wild type CRISPR/Cas12a endonuclease system and may display reduced off-target editing activity when compared to wild type CRISPR/Cas12a endonuclease system.
In another embodiment the CRISPR/Cas12a endonuclease system of the present invention displays maintained on-target editing activity relative to a wild type CRISPR/Cas endonuclease system and may display reduced off-target editing activity when compared to wild type CRISPR/Cas endonuclease systems. In another aspect the CRISPR/Cas12a endonuclease system of the present invention displays maintained on-target editing activity relative to a wild type CRISPR/Cas12a endonuclease system and may display reduced off-target editing activity when compared to wild type CRISPR/Cas12a endonuclease system.
The methods and compositions of the invention described herein provide mutant LbCas12a nucleic acids and polypeptides for use in a CRISPR/Cas12a system. The present invention describes novel Cas12a mutants that reduce off-target editing activity to low levels while maintaining high on-target editing activity relative to the wild-type protein even when delivered as an RNP complex. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Cas12a provides a useful complement to Cas9 by expanding the range of PAM sequences that can be targeted from GC-Rich areas (Cas9) to AT-rich areas of the genome (Cas12a), thereby expanding the range of sequences that can be modified using CRISPR genome engineering methods. In addition to having a T-rich PAM site, another advantage of the Cas12a system as compared with Cas9 is the use of a single short RNA molecule.
In a first embodiment an isolated mutant Cas12a protein is provided. The isolated mutant Cas12a protein is active in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein endonuclease system (“CRISPR/Cas12a endonuclease system”). The CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system. In another aspect the Cas12a protein is isolated from Lachnospiraceae bacterium ND2006 (Lb). Preferred single mutant Cas12a proteins include substitution mutations into the WT-LbCas12a introduced at the following positions: N527, D559, and E795.
Exemplarily single mutant Cas12a proteins include the following specific mutations introduced into the WT-LbCas12a: N527R, D559P, and E795L. Exemplary single mutant Cas12a proteins include at least one-member selected form the group consisting of SEQ ID NO: 3-9. Additional substitution mutations can be included in the amino acid backgrounds of the single mutant Cas12a protein amino acid sequences, provided that the resultant Cas12a protein is active as a CRISPR/Cas12a endonuclease system, wherein the resultant CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system.
Preferred multi-substitution mutant Cas12a proteins include mutations in the WT-LbCas12a introduced to at least two of the following positions: N527/D559, D559/E795, N527/E795, and N527/D559/E795. Exemplary multi-substitution mutant Cas12a proteins include mutations in the WT-LbCas12a selected from the following amino acid mutations: N527R/D559P, D559P/E795L, N527R/E795L and N527R/D559P/E795L.
Exemplary multi substitution mutant Cas12a proteins include at least one member selected from the group consisting of SEQ ID NO: 3-9. Additional substitution mutations can be included in the amino acid backgrounds of the single mutant Cas12a protein amino acid sequences, provided that the resultant Cas12a protein is active as a CRISPR/Cas12a endonuclease system, wherein the resultant CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system.
In second embodiment, an isolated ribonucleoprotein complex is provided. The RNP includes mutant Cas12a protein and a crRNA complex. In one respect the crRNA includes an Alt-R® crRNA (Integrated DNA Technologies, Inc. (Coralville, IA, (US)) directed against a specific editing target site for a given locus. Preferred mutant Cas9 proteins include those as described above.
In another embodiment, an isolated nucleic acid encoding a mutant LbCas12a protein is provided. Preferred isolated nucleic acids encode mutant LbCas12a proteins as described above. Exemplary isolated nucleic acids encoding mutant LbCas12a proteins can be readily generated from a nucleic acid encoding the wild-type LbCas12a protein using recombinant DNA procedures or chemical synthesis methods. Preferred nucleic acids for this purpose include those optimized for expression of the LbCas12a proteins in bacteria, (e.g., E. coli) or mammalian (e.g., human) cells. Exemplary codon-optimized nucleic acids for expressing WT-LbCas12a in E. coli and human cells includes SEQ ID NO: 1. Moreover, the present invention contemplates fusion proteins of WT-Cas12a and mutant LbCas12a, wherein the coding sequences of WT-Cas12a and mutant LbCas12a are fused to amino acid sequences encoding for nuclear localization (“NLS”) of the fusion protein in eukaryotic cells or amino acid sequences to facilitate purification of the proteins.
In a further embodiment, the isolated nucleic acid includes mRNA encoding one of the aforementioned mutant LbCas12a proteins. In a second respect, the isolated nucleic acid includes DNA encoding a gene for one of the aforementioned mutant LbCas12a proteins. A preferred DNA includes a vector that encodes a gene encoding for a mutant LbCas12a protein. Such delivery methods include plasmid and various viral delivery vectors as are well known to those with skill in the art. The mutant LbCas12a protein can also be stably transformed into cells using suitable expression vectors to produce a cell line that constitutively or inducibly expresses the mutant LbCas12a. The aforementioned methods can also be applied to embryos to product progeny animals that constitutively or inducibly expresses the mutant LbCas12a.
In another embodiment a CRISPR/Cas12a endonuclease systems is provided.
The CRISPR/Cas12a endonuclease system includes a mutant LbCas12a protein. Preferred mutant LbCas12a proteins include those describe above. In one aspect, the CRISPR/Cas12a endonuclease system is encoded by a DNA expression vector. In one embodiment, the DNA expression vector is selected from a bacterial expression vector or a eukaryotic expression vector. In another aspect the CRISPR/Cas12a endonuclease system comprises a ribonucleoprotein complex comprising a mutant LbCas12a protein and a crRNA.
In a further embodiment, a method of performing gene editing having increased on-target editing activity is provided. The method includes the step of contacting a candidate editing target site locus with an active CRISPR/Cas12a endonuclease system having a mutant LbCas12a protein. In one aspect, the method includes a single mutant LbCas12a protein having mutations in the WT-LbCas12a introduced at one of the following positions: N527, D559, and E795. Exemplary single mutant LbCas12a proteins include the following specific mutations introduced into the WT-LbCas 12a: N527R, D559P, and E795L. Exemplary single mutant LbCas12a proteins include at least one member selected form the group consisting of SEQ ID NO: 3-9. Additional substitution mutations can be included in the amino acid backgrounds of the single mutant LbCas12a protein amino acid sequences, provided that the resultant LbCas12a protein is active as a CRISPR/Cas12a endonuclease system, wherein the resultant CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system.
In another embodiment, the method includes a multi-substitution mutant LbCas12a proteins include mutations in the WT-LbCas12a introduced to at least two of the following positions: N527/D559, D559/E795, N527/E795, and N527/D559/E795. Exemplary multi-substitution mutant Cas12a proteins include mutations in the WT-LbCas12a selected from the following amino acid mutations: N527R/D559P, D559P/E795L, N527R/E795L and N527R/D559P/E795L. Exemplary multi substitution mutant LbCas12a proteins include at least one member selected from the group consisting of SEQ ID NO: 3-9. Additional substitution mutations can be included in the amino acid backgrounds of the single mutant LbCas12a protein amino acid sequences, provided that the resultant LbCas12a protein is active as a CRISPR/Cas12a endonuclease system, wherein the resultant CRISPR/Cas12a endonuclease system displays maintained on-target editing activity relative to a wild-type CRISPR/Cas12a endonuclease system.
The applications of Cas12a and LbCas12a based tools are many and varied. The applications include, but are not limited to: plant gene editing, yeast gene editing, mammalian gene editing, editing of cells in the organs of live animals, editing of embryos, rapid generation of knockout/knock-in animal lines, generating an animal model of disease state, correcting a disease state, inserting a reporter gene, and whole genome functional screening.
The list below shows different wild type (WT) and mutant Cas12a nucleases described in present invention. It will be appreciated by one with skill in the art that many different DNA sequences can encode/express the same amino acid (AA) sequence since in many cases more than one codon can encode the same amino acid. The DNA sequences shown below only serve as example and other DNA sequences that encode the same protein (e.g., same amino acid sequence) are contemplated. It is further appreciated that additional features, elements or tags may be added to said sequences, such as NLS domains and the like. Examples are shown for WT LbCas12a (Cpf1), WT AsCas12a, and mutant N527R LbCas12a, mutant D559P LbCas12a, mutant E759L LbCas12a, double mutant N527R/D559P LbCas12a, double mutant N527R/E795L LbCas12a, double mutant D559P/E795L LbCas12a, triple mutant N527R/D559P/E795L LbCas12a, and double mutant M537R/F870L AsCas12a. For LbCas12a and AsCas12a mutants only the amino acid and DNA sequences are provided, but it is contemplated that NLS domains and His-tag domains may be added to facilitate use in producing recombinant proteins for use in mammalian cells.
Overexpression and Purification of LbCas12a Mutants in E. coli Cells
This example demonstrates the over expression and purification of seven Cas12a mutants, N527R, D559P, E795L, N527R/D559P, D559P/E795L, N527R/E795L and N527R/D559P/E795L. The LbCas12a mutants were introduced by site-directed mutagenesis, using standard PCR conditions and primers (Table 1). After transformation into E. coli BL21 (DE3) cells, a colony with the appropriate strain was used to inoculate TB media with kanamycin (0.05 mg/mL) and grown at 37° C. until an OD of approximately 0.9 was reached, then the flask was cooled to 18° C. for 30 minutes. The addition of 1 M IPTG (500 μL) was used to induce protein expression, followed by growth at 18° C. for 19 hours. Cells were harvested and the cell pellet was re-suspended and lysed on an Avestin Emulsiflex C3 pre-chilled to 4° C. at 15-20 kpsi with three passes. The lysate was centrifuged at 16,000×g for 20 minutes at 4° C. to remove cell debris
The cleared lysate was put over a HisTrap HP column. The procedure consisted of equilibrating the resin with His-Bind buffer (20 mM NaPO4 pH 6.8, 0.5 M NaCl, 10 mM imidazole, 5% glycerol), followed by sample loading. The sample was washed with His-Bind buffer, followed by an additional standard wash and a 10% “B” wash consisting of 10% His-Elution buffer (10 mM NaPO4 pH 6.8, 250 mM NaCl, 150 mM imidazole, 5% glycerol). Finally, the sample was eluted using His-Elution buffer. The LbCas12a mutants were then put over a HiTrap Heparin HP column. The procedure consisted of equilibrating the resin with the Heparin-Bind buffer (20 mM NaPO4 pH 6.8, 250 mM NaCl, 10% glycerol), followed by sample loading. The sample was then washed with Heparin-Bind buffer, followed by a 5% “B” wash consisting of 5% Heparin Elution buffer (10 mM NaPO4 pH 6.8, 1 M NaCl, 10% glycerol). Finally, the purified protein was eluted using Heparin Elution buffer (10 mM NaPO4, pH 6.8, 1 M NaCl, 10% glycerol). Finally, the purified protein was eluted using Heparin.
The purified LbCas12a mutants were concentrated to approximately 10 mg/mL and stored at −20° C. in storage buffer (25 mM Tris-HCl PH 7.4, 0.3 M NaCl). 1 mM EDTA, 1 mM DTT, 50% glycerol).
Novel LbCas12a Substitution Mutants Enhance the Cleavage Activity in a Human Cell Line Based Activity Assay when Delivered Into Human Cells Via Ribonucleoprotein Complex.
The following example demonstrates the ability of LbCas12a mutants to improve genome editing efficiency when delivered as an RNP complex. The example demonstrates the ability of LbCas12a mutants to show comparable genome editing efficiency when delivered at a high dose and increased genome editing efficiency when delivered at a low dose by ribonucleoprotein (RNP) complex into human cells with electroporation transfection.
The RNP complex was formed by incubating purified LbCas12a and sgRNA (Table 2, entries 3 and 9) at a ratio of 1:1.2 in PBS buffer for 10 minutes at room temperature. The RNP complexes (5 μM final dose) were transfected into HEK293 immortalized human cells using a Lonza 4D-Nucleofector™ and Amaxa® 96-well Shuttle Device with Alt-R® Cpf1 Electroporation Enhancer (3 μM, Integrated DNA Technologies). The experiments were performed in biological triplicate and after 48 hours at 37° C., adherent cells were lysed with QuickExtract™ DNA extraction solution (50 μL).
Crude lysates were incubated at 65° C. for 15 minutes, followed by heat inactivation at 98° C. for 3 minutes. Crude genomic DNA was diluted 5-fold in TE buffer and used as PCR template. PCR (primers listed in Table 3) was used to amplify 1.2 kbp fragments of the HPRT loci using Q5® DNA Polymerase (New England Biolabs) and the following parameters: 98° C. for 30 sec, followed by 98° C. for 10 sec, 65° C. for 15 sec and 72° C. for 1 min which was repeated 24 times, followed by a final extension at 72° C. for 2 min. Heteroduplexes were formed by the addition of NEBuffer 2 and initially heating to 95° C. for 10 min with a slow cool down to room temperature. The heteroduplexes were then cleaved by 2 U of T7 Endonuclease I (New England Biolabs) for 1 hour at 37° C. The cleavage products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical).
The endonuclease activity of wild type and mutant LbCas12a in HEK293 human cells are described in
The dose response was achieved as described above, reducing the amount of RNP by 2-fold increments (with and without Alt-R® Electroporation Enhancer) and beginning with a 2 μM dose. The results are shown in
The final dose response in this example was set up as described above, reducing the amount of RNP even further by 5-fold increments, starting with a 2 μM dose. The RNP was formed using sgRNA 2 and 8 for the LbCas12a RNP and 14 and 20 for the AsCas12a RNP (Table 1). The results are shown in
Single LbCas12a Substitution Mutant Enhances the Cleavage Activity in a Human Cell Line Based Activity Assay when Delivered Into Human Cells Via Ribonucleoprotein Complex at Low Doses.
The following example demonstrates the ability of mutant E795L LbCas12a to show increased genome editing efficiency when delivered at low doses by RNP complex into human cells with electroporation transfection. That this invention increases genome editing efficiency when wild-type or mutant Cas12a is delivered into human cells as an RNP complex.
The RNP complex was formed by incubating purified Cas12a and sgRNA (Table 2, entries 1-12 for the LbCas12a RNP and entries 13-24 for the AsCas12a RNP) at a ratio of 1:1.2 in PBS buffer for 10 minutes at room temperature. The RNP complexes (1, 0.22, 0.05 and 0.01 μM final doses) were transfected into HEK293 immortalized human cells using a Lonza 4D-Nucleofector™ and Amaxa® 96-well Shuttle Device with Alt-R® Cpf1 Electroporation Enhancer (3 μM, Integrated DNA Technologies). The experiments were performed in biological duplicate and after 48 hours at 37° C., adherent cells were lysed with QuickExtract™ DNA extraction solution (50 μL).
Crude lysates were incubated at 65° C. for 15 minutes, followed by heat inactivation at 98° C. for 3 minutes. Crude genomic DNA was diluted 15-fold in TE buffer and used as PCR template. PCR (primers listed in Table 3) was used to amplify 1.2 kbp fragments of the HPRT loci using Q5® DNA Polymerase (New England Biolabs) and the following parameters: 98° C. for 30 sec, followed by 98° C. for 10 sec, 65° C. for 15 sec and 72° C. for 1 min which was repeated 24 times, followed by a final extension at 72° C. for 2 min. Heteroduplexes were formed by the addition of NEBuffer 2 and initially heating to 95° C. for 10 min with a slow cool down to room temperature. The heteroduplexes were then cleaved by 2 U of T7 Endonuclease I (New England Biolabs) for 1 hour at 37° C. The cleavage products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical).
The endonuclease activity of wild type and mutant LbCas12a as compared to wild type and mutant AsCas12a in HEK293 human cells are described in
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but no limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention, and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The term “wild-type LbCas12a” (“wild-type Lb enzyme” or “WT-LbCas12a”) encompasses a protein having the identical amino acid sequence of the naturally-occurring Lachnospiraceae bacterium ND2006 Cas12a (e.g., SEQ ID NO: 2) and that has biochemical and biological activity when combined with a suitable crRNA to form and active CRISPR/Cas12a endonuclease system. The term “wild-type AsCas12a” (“wild-type as enzyme” or “WT-AsCas12a”) encompasses a protein having the identical amino acid sequence of the naturally-occurring Acidaminococcus sp. BV3L6 Cas12a (e.g., SEQ ID NO: 18) and that has biochemical and biological activity when combined with a suitable crRNA to form and active CRISPR/Cas12a endonuclease system.
The term “mutant LbCas12a protein” encompasses protein forms having a different amino acid sequence form the wild-type Lachnospiraceae bacterium ND2006 Cas12a and that have biochemical and biological activity with combined with a suitable crRNA to form an active CRISPR-Cas12a endonuclease system. This includes orthologs and Cas12a variants having different amino acid sequences form the wild-type Lachnospiraceae bacterium ND2006 Cas12a.
The term “polypeptide” refers to any linear or branched peptide comprising more than one amino acid. Polypeptide includes protein or fragment thereof or fusion thereof, provided such protein, fragment or fusion retains a useful biochemical or biological activity.
Fusion proteins typically include extra amino acid information that is not native to the protein to which the extra amino acid information is covalently attached. Such extra amino acid information may include tags that enable purification or identification of the fusion protein. Such extra amino acid information may include peptides that enable the fusion proteins to be transported into cells and/or transported to specific locations within cells. Examples of tags for these purposes include the following: AviTag, which is a peptide allowing biotinylation by the enzyme BirA so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE) (SEQ ID NO: 53); Calmodulin-tag, which is a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 54); polyglutamate tag, which is a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE) (SEQ ID NO: 55); E-tag, which is a peptide recognized by an antibody (GAPVPYPDPLEPR) (SEQ ID NO: 56); FLAG-tag, which is a peptide recognized by an antibody (DYKDDDDK) (SEQ ID NO: 57); HA-tag, which is a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA) (SEQ ID NO: 58); His-tag, which is typically 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH) (SEQ ID NO: 59); Myc-tag, which is a peptide derived from c-myc recognized by an antibody (EQKLISEEDL) (SEQ ID NO: 60); NE-tag, which is a novel 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES) (SEQ ID NO: 61) recognized by a monoclonal IgG1 antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, which is a peptide derived from Ribonuclease A (KETAAAKFERQHMDS) (SEQ ID NO: 62); SBP-tag, which a peptide which binds is to streptavidin; (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 63); Softag 1, which is intended for mammalian expression (SLAELLNAGLGGS) (SEQ ID NO: 64); Softag 3, which is intended for prokaryotic expression (TQDPSRVG) (SEQ ID NO: 65); Strep-tag, which is a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK) (SEQ ID NO: 66); TC tag, which is a tetracysteine tag that is recognized by FIAsH and ReAsH biarsenical compounds (CCPGCC) (SEQ ID NO: 67); V5 tag, which is a peptide recognized by an antibody (GKPIPNPLLGLDST) (SEQ ID NO: 68); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK) (SEQ ID NO: 69); Xpress tag (DLYDDDDK) (SEQ ID NO: 70); Isopeptag, which is a peptide which binds covalently to pilin-C protein (TDKDMTITFTNKKDAE) (SEQ ID NO: 71); SpyTag, which is a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK) (SEQ ID NO: 72); SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK) (SEQ ID NO: 73); BCCP (Biotin Carboxyl Carrier Protein), which is a protein domain biotinylated by BirA to enable recognition by streptavidin; Glutathione-S-transferase-tag, which is a protein that binds to immobilized glutathione; Green fluorescent protein-tag, which is a protein which is spontaneously fluorescent and can be bound by antibodies; HaloTag, which is a mutated bacterial haloalkane dehalogenase that covalently attaches to a reactive haloalkane substrate to allow attachment to a wide variety of substrates; Maltose binding protein-tag, a protein which binds to amylose agarose; Nustag; Thioredoxin-tag; and Fc-tag, derived from immunoglobulin Fc domain, which allows dimerization and solubilization and can be used for purification on Protein-A Sepharose. Nuclear localization signals (NLS), such as those obtained from SV40, allow for proteins to be transported to the nucleus immediately upon entering the cell. Given that the native Cas9 protein is bacterial in origin and therefore does not naturally comprise a NLS motif, addition of one or more NLS motifs to the recombinant Cas9 protein is expected to show improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate resides in the nucleus. One skilled in the art would appreciate these various fusion tag technologies, as well as how to make and use fusion proteins that include them.
This application is a divisional of U.S. patent application Ser. No. 17/430,013, filed Aug. 11, 2021, which is a U.S. national stage entry of International Patent Application No. PCT/US2020/019168, filed on Feb. 21, 2020, which claims the benefit to U.S. Provisional Patent Application No. 62/808,984, filed on Feb. 22, 2019, each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62808984 | Feb 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17430013 | Aug 2021 | US |
| Child | 19061780 | US |
