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Compositions and methods of engineered CRISPR-Cas9 systems using split-nexus … — Caribou Biosciences, Inc. (US11111506B2)

Caribou Biosciences, Inc. · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US11111506B2, Caribou Biosciences, Inc., Paul Daniel Donohoue, en, 2021

ABSTRACT

Abstract

The present specification discloses engineered Type II CRISPR-Cas9 systems comprising split-nexus Cas9-associated polynucleotides (sn-casPNs), including systems comprising three split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN/sn3-casPN) and systems comprising two split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN). Together with a Cas9 protein, the sn-casPNs facilitate site-specific modifications, including cleavage and mutagenesis, of a target polynucleotide in vitro and in vivo. Furthermore, the engineered Type II CRISPR-Cas9 systems comprising sn-casPNs are useful in methods of regulating expression of a target nucleic acid. Methods are described herein for the creation of a variety of engineered Type II CRISPR-Cas9 systems comprising two or more sn-casPNs. Polynucleotide sequences, expression cassettes, vectors, compositions, and kits for carrying out a variety of methods are also described. Furthermore, the present specification provides genetically modified cells, compositions of modified cells, transgenic organisms, pharmaceutical compositions, as well as a variety of compositions and methods involving the engineered Type II CRISPR-Cas9 systems.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No. 15/665,201, filed 31 Jul. 2017, now U.S. Pat. No. 9,970,027, issued 15 May 2018, which is a Continuation of U.S. patent application Ser. No. 15/339,633, filed 31 Oct. 2016, now U.S. Pat. No. 9,745,600, issued 29 Aug. 2017, which is a Continuation of U.S. patent application Ser. No. 14/835,675, filed 25 Aug. 2015, now U.S. Pat. No. 9,580,727, issued 28 Feb. 2017, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/202,715, filed 7 Aug. 2015, and U.S. Provisional Patent Application Ser. No. 62/209,334, filed 24 Aug. 2015, the contents of which are herein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

SEQUENCE LISTING

The present application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on 26 Apr. 2018, is named CBI017-14_ST25.txt and is 20 kb in size.

TECHNICAL FIELD

The present invention relates to engineered Type II CRISPR-Cas9 systems.

BACKGROUND OF THE INVENTION

Genome engineering includes altering the genome by deleting, inserting, mutating, or substituting specific nucleic acid sequences. The alteration can be gene or location specific. Genome engineering can use nucleases to cut DNA, thereby generating a site for alteration. In certain cases, the cleavage can introduce double-stranded breaks in the target DNA. Double-stranded breaks can be repaired, e.g., by endogenous non-homologous end joining (NHEJ) or homology-directed repair (HDR). HDR relies on the presence of a template for repair. In some examples of genome engineering, a donor polynucleotide, or portion thereof, can be inserted into the break.

Clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins constitute the CRISPR-Cas system. This system provides adaptive immunity against foreign DNA in bacteria (Barrangou, R., et al., âCRISPR provides acquired resistance against viruses in prokaryotes,â Science 315, 1709-1712 (2007); Makarova, K. S., et al., âEvolution and classification of the CRISPR-Cas systems,â Nat Rev Microbiol 9, 467-477 (2011); Garneau, J. E., et al., âThe CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA,â Nature 468, 67-71 (2010); Sapranauskas, R., et al., âThe Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli ,â Nucleic Acids Res 39, 9275-9282 (2011)). The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner (Gasiunas, G., et al., âCas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,â Proc Natl Acad Sci USA 109, E2579-E2586 (2012); Jinek, M., et al., âA programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,â Science 337, 816-821 (2012); Sternberg, S. H., et al., âDNA interrogation by the CRISPR RNA-guided endonuclease Cas9,â Nature 507, 62 (2014); Deltcheva, E., et al., âCRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,â Nature 471, 602-607 (2011)), and has been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L., et al., âMultiplex genome engineering using CRISPR/Cas systems,â Science 339, 819-823 (2013); Jiang, W., et al., âRNA-guided editing of bacterial genomes using CRISPR-Cas systems,â Nat. Biotechnol. 31, 233-239 (2013); Sander, J. D. & Joung, J. K., âCRISPR-Cas systems for editing, regulating and targeting genomes,â Nature Biotechnol. 32, 347-355. (2014)).

Jinek, M., et al., (âA programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,â Science 337(6096):816-21 (2012)) showed that in a subset of CRISPR-associated (Cas) systems the mature CRISPR (crRNA) that is base paired to trans-activating crRNA (tracrRNA) forms a two-part RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded breaks in target DNA. At sites complementary to the crRNA-guide (spacer) sequence, the Cas9 HNH nuclease domain cleaves the complementary strand and the Cas9 RuvC-like domain cleaves the non-complementary strand. Dual crRNA/tracrRNA molecules were engineered into single-chain crRNA/tracrRNA molecules. These single-chain crRNA/tracrRNA directed target sequence-specific Cas9 double-strand DNA cleavage.

Jinek, M., et al., designed two versions of single-chain crRNA/tracrRNA containing a target recognition sequence (spacer) at the 5â² end followed by a hairpin structure retaining the base-pairing interactions that normally occur between the tracrRNA and the crRNA (see FIG. 5B of Jinek, M., et al.). For each single-chain crRNA/tracrRNA, the 3â² end of crRNA was covalently attached to the 5â² end of tracrRNA. In cleavage assays using plasmid DNA, Jinek, M., et al., observed that a 3â² truncated single-chain crRNA/tracrRNA did not cleave target DNA as efficiently in the assay as a longer single-chain crRNA/tracrRNA that was not truncated at the 3â² end (see FIG. 5B and FIGS. 14 A, B, and C of Jinek, M., et al.). These data confirmed that the â5 to 12 positions beyond the tracrRNA:crRNA base-pairing interaction are important for efficient Cas9 binding and/or target recognitionâ (Jinek, M., et al., Science 337(6096):820 (2012)).

Briner, A., et al., (âGuide RNA Functional Modules Direct Cas9 Activity and Orthogonality,â Molecular Cell 56(2), 2014, Pages 333-339) elucidated the molecular basis of selective Cas9/guide-RNA interactions by identifying and characterizing distinct sequence and structural modules within guide RNAs that direct Cas9 endonuclease activity and define orthogonality. They established six modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) across forty-one systems from three distinct Cas9 families. The six identified modules are the spacer, the lower stem, the bulge, the upper stem, the nexus hairpin, and 3â² hairpins. These modules are illustrated with reference to an sgRNA in FIG. 2 .

Using the sgRNA/Cas9 system from Streptococcus pyogenes , Briner, A., et al., showed that a bulge within the sgRNA is structurally necessary for DNA cleavage both in vitro and in vivo, whereas sequence substitutions are tolerated in other regions. Furthermore, expendable features can be removed to generate functional miniature sgRNAs. They also identified a conserved module ânamed the nexus; this feature exhibits sequence and structural features important for cleavageâ (Briner, A., et al., page 2). They stated that this module, the nexus, is ânecessary for DNA cleavageâ (Briner, A., et al., Summary). The nexus hairpin confers activity to its cognate Cas9. The location of this nexus hairpin corresponds to the 5 to 12 positions beyond the tracrRNA:crRNA base-pairing interaction that are important for efficient Cas9 binding and/or target recognition as identified by Jinek, M., et al. (see above).

Briner, A., et al., showed that the general nexus hairpin shape with a GC-rich stem and an offset uracil was shared between the two Streptococcus families. In contrast, the idiosyncratic double stem of the nexus hairpin was unique to, and ubiquitous in, Lactobacillus systems. Some bases within the nexus hairpin were strictly conserved even between distinct families, including A52 and C55, further highlighting the important role of this module. In the crystal structure of SpyCas9 A52 interacts with the backbone of residues 1103-1107 close to the 5â² end of the target strand in the in the crystal structure of SpyCas9, suggesting that the interaction of the nexus hairpin with the protein backbone may be required for protospacer-adjacent motif (PAM) binding.

Wright, A. V., et al., (âRational design of a split-Cas9 enzyme complex,â PNAS 112(10), 2015, pages 2984-2989) determined the RNA molecular determinants of sgRNA motifs that promote heterodimerization of the Î±-helical and nuclease lobes to form a ternary complex. Crystal structures of sgRNA/DNA-bound Cas9 showed that the spacer and the stem-loop motifs (i.e., the lower stem, the bulge, and the upper stem modules described by Briner, A., et al.) at the 5â² end of the sgRNA primarily contact the Î±-helical lobe, whereas the two hairpins (i.e., the hairpins module described by Briner, A., et al.) at the 3â² end bind the outside face of the nuclease lobe. They noted that âthe nexus motif, recently shown to be critical for activityâ (Wright, A. V., et al., page 2986, col. 1), occupies a central position between the lobes and forms extensive interactions with the bridge helix. Based on this interaction profile, Wright, et al., generated a full-length sgRNA and two shorter sgRNA constructs that were selectively truncated from either the 5â² or 3â² end (no modifications were made to the critical nexus hairpin) and determined their affinities for wild-type Cas9, the individual Î±-helical and nuclease lobes, and split-Cas9.

Contrary to the above-described teachings of the prior art, experiments performed in support of the present invention unexpectedly demonstrated that Cas9 functions (e.g., binding and cutting double-strand DNA) are supported by guide RNAs having a split nexus, as well as guide RNAs having modifications of the split nexus.

Results presented in the present specification open new design and engineering avenues for CRISPR technologies and set the stage for the development of next-generation CRISPR-based technologies.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to engineered Type II CRISPR-Cas9 system wherein at least two polynucleotides are necessary to form a nexus stem element.

In one aspect, the present invention relates to an engineered Type II CRISPR-Cas9 system comprising three polynucleotides capable of forming a complex with a Cas9 protein to cause the Cas9 protein to bind a first DNA sequence comprising a DNA target sequence preferentially relative to a second DNA sequence without the DNA target binding sequence. At least two of the three polynucleotides are necessary to form a nexus stem element. In some embodiments, the engineered Type II CRISPR-Cas9 system further comprises a Cas9 protein or a DNA sequence encoding a Cas9 protein. In additional embodiments, the present invention relates to the three polynucleotides in complex with a Cas9 protein.

In one embodiment, an engineered Type II CRISPR-Cas9 system of the present invention comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide that are separate polynucleotides each having a 5â² end and a 3â² end.

The first polynucleotide comprising in a 5â² to 3â² direction a first stem element nucleotide sequence I and a nexus stem element nucleotide sequence I. The second polynucleotide comprising a nexus stem element nucleotide sequence II, wherein the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II are capable of forming the nexus stem element by base-pair hydrogen bonding between the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II. The third polynucleotide comprising in a 5â² to 3â² direction a DNA target binding sequence and a first stem element nucleotide sequence II, wherein the first stem element nucleotide sequence I and the first stem element nucleotide sequence II are capable of forming a first stem element by base-pair hydrogen bonding between the first stem element nucleotide sequence I and the first stem element nucleotide sequence II.

In another embodiment, an engineered Type II CRISPR-Cas9 system of the present invention comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide that are separate polynucleotides each having a 5â² end and a 3â² end. The first polynucleotide comprising in a 5â² to 3â² direction an upper stem element nucleotide sequence I, a bulge element nucleotide sequence I, a lower stem element nucleotide sequence I, and a nexus stem element nucleotide sequence I. The second polynucleotide comprising a nexus stem element nucleotide sequence II, wherein the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II are capable of forming the nexus stem element by base-pair hydrogen bonding between the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II. The third polynucleotide comprising in a 5â² to 3â² direction a DNA target binding sequence, a lower stem element nucleotide sequence II, a bulge element nucleotide sequence II, and an upper stem element nucleotide sequence II, wherein the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are capable of forming an upper stem element by base-pair hydrogen bonding between the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II, and the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II are capable of forming a lower stem element by base-pair hydrogen bonding between the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II.

In further embodiments the second polynucleotide comprises first and/or second adjunct polynucleotides. The second polynucleotide can further comprise in a 5â² to 3â² direction the nexus stem element nucleotide sequence II and a second stem element nucleotide sequence I, and a first adjunct polynucleotide that comprises a second stem element nucleotide sequence II. The second stem element nucleotide sequence I and the second stem element nucleotide sequence II are capable of forming a second stem element by base-pair hydrogen bonding between the second stem element nucleotide sequence I and the second stem element nucleotide sequence II. In some embodiments, the first adjunct polynucleotide further comprises in a 5â² to 3â² direction a loop element nucleotide sequence and the second stem element nucleotide sequence II, wherein 5â² end of the loop element nucleotide sequence is covalently bonded to the 3â² end of the second stem element nucleotide sequence I, thus forming a hairpin. In yet further embodiments, the first adjunct polynucleotide comprises in a 5â² to 3â² direction the second stem element nucleotide sequence II and a third stem element nucleotide sequence I, and a second adjunct polynucleotide comprises in a 5â² to 3â² direction a third stem element nucleotide sequence II. The third stem element nucleotide sequence I and the third stem element nucleotide sequence II are capable of forming a third stem element by base-pair hydrogen bonding between the third stem element nucleotide sequence I and third stem element nucleotide sequence II. In some embodiments the second adjunct polynucleotide further comprises in a 5â² to 3â² direction a loop element nucleotide sequence and the third stem element nucleotide sequence II, wherein 5â² end of the loop element nucleotide sequence is covalently bonded to the 3â² end of the third stem element nucleotide sequence I.

Additional embodiments of the present invention include the first polynucleotide further comprising a first auxiliary polynucleotide 3â² adjacent the nexus stem element nucleotide sequence I, the second polynucleotide further comprising a second auxiliary polynucleotide 5â² adjacent the nexus stem element nucleotide sequence II, or both the first polynucleotide and the second polynucleotide each comprising an auxiliary sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

SEQUENCE LISTING

TECHNICAL FIELD

The present invention relates to engineered Type II CRISPR-Cas9 systems.

BACKGROUND OF THE INVENTION

Results presented in the present specification open new design and engineering avenues for CRISPR technologies and set the stage for the development of next-generation CRISPR-based technologies.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to engineered Type II CRISPR-Cas9 system wherein at least two polynucleotides are necessary to form a nexus stem element.

In some embodiments of the present invention, the first auxiliary polynucleotide comprises an effector binding element nucleotide sequence I, and the second auxiliary polynucleotide comprises an effector binding element nucleotide sequence II. The effector binding element nucleotide sequence I and the effector binding element nucleotide sequence II are capable of forming an effector binding element by base-pair hydrogen bonding between the effector binding element nucleotide sequence I and the effector binding element nucleotide sequence I. The effector binding element can be, for example, a double-stranded RNA and the effector protein is a double-stranded RNA binding protein capable of binding the effector binding element. In selected embodiments the effector protein is a catalytically inactive variant of a protein selected from the group consisting of Cas5, Cas6, and Csy4.

In additional embodiments, the first auxiliary polynucleotide further comprises in a 5â² to 3â² direction a linker element nucleotide sequence I and the effector binding element nucleotide sequence I, and the second auxiliary polynucleotide comprises in a 5â² to 3â² direction the effector binding element nucleotide sequence II and a linker element nucleotide sequence II. The linker element nucleotide sequence I and the linker element nucleotide sequence II are capable of forming a linker element by base-pair hydrogen bonding between the effector binding element nucleotide sequence I and the effector binding element nucleotide sequence I.

In yet further embodiments, the first auxiliary polynucleotide, the second auxiliary polynucleotide, or both the first auxiliary polynucleotide and the second auxiliary polynucleotide each comprises a hairpin. Furthermore, the first auxiliary polynucleotide can further comprises in a 5â² to 3â² direction a linker element nucleotide sequence I and the hairpin, the second auxiliary polynucleotide comprises in a 5â² to 3â² direction the hairpin and a linker element nucleotide sequence II, or both the first auxiliary polynucleotide comprises in a 5â² to 3â² direction a linker element nucleotide sequence I and the hairpin and the second auxiliary polynucleotide comprises in a 5â² to 3â² direction the hairpin and a linker element nucleotide sequence II. The linker element nucleotide sequence I and the linker element nucleotide sequence II are capable of forming linker element by base-pair hydrogen bonding between the effector binding element nucleotide sequence I and the effector binding element nucleotide sequence I.

In another aspect an engineered Type II CRISPR-Cas9 system of the present invention comprises three polynucleotides. A first polynucleotide comprises in a 5â² to 3â² direction an upper stem element nucleotide sequence I, a bulge element nucleotide sequence I, a lower stem element nucleotide sequence I, and a nexus stem element nucleotide sequence I. A second polynucleotide comprises in a 5â² to 3â² direction a nexus stem element nucleotide sequence II, a second stem element comprising a hairpin, and a third stem element comprising a hairpin. The nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II are capable of forming the nexus stem element by base-pair hydrogen bonding between the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II. A third polynucleotide comprises in a 5â² to 3â² direction a DNA target binding sequence, a lower stem element nucleotide sequence II, a bulge element nucleotide sequence II, and an upper stem element nucleotide sequence II. The upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II are capable of forming an upper stem element by base-pair hydrogen bonding between the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II, and the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II are capable of forming a lower stem element by base-pair hydrogen bonding between the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II. The engineered Type II CRISPR-Cas9 system can further comprise a Cas9 protein or a DNA sequence encoding a Cas9 protein.

In some embodiments of this aspect of the present invention the first polynucleotide further comprises a first auxiliary polynucleotide 3â² adjacent the nexus stem element nucleotide sequence I, and the second polynucleotide further comprises a second auxiliary polynucleotide 5â² adjacent the nexus stem element nucleotide sequence II.

These aspects and other embodiments of the present invention using the sn-casPNs/Cas9 protein systems of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B present illustrative examples of dual guide Type II CRISPR-Cas9 associated RNAs.

FIG. 2 shows another example of a CRISPR-Cas9 associated RNA.

FIG. 3A , FIG. 3B , FIG. 3C , FIG. 3D , FIG. 3E , FIG. 3F , FIG. 3G , and FIG. 3H present a variety of polynucleotides of the engineered Type II CRISPR-Cas9 systems of the present invention.

FIG. 4A and FIG. 4B present further modifications of the polynucleotides described in FIG. 3A through FIG. 3H .

FIG. 5A , FIG. 5B , and FIG. 5C relate to structural information for an embodiment of a sn1-casRNA/sn2-casRNA/Cas9 protein complex, wherein the sn1-casRNA, sn2-casRNA correspond to sn1-casPN and sn2-casPN of FIG. 3B .

FIG. 6A , FIG. 6B , and FIG. 6C illustrate an example of a split-nexus Cas9-associated two polynucleotide system and its association with a Cas9 protein.

FIG. 7A and FIG. 7B illustrate an example of a split-nexus Cas9-associated two polynucleotide system and its association with a Cas9 protein.

FIG. 8 presents the results of the Cas9 cleavage assay using the AAVS-1 target double-stranded DNA.

FIG. 9 presents the results of the Cas9 cleavage assay using the Csy4* protein to enhance the cleavage activity of the sn-casRNAs comprising an additional Csy4 RNA binding sequence.

FIG. 10 presents the result of the Cas9 cleavage assay using sn1-casRNAs EX2 and sn2-casRNA EX2 .

FIG. 11 presents the results of Cas9 cleavage assays.

FIG. 12 presents examples of putative split nexus arrangements of known tracrRNA sequences from certain bacterial species.

FIG. 13 is an oligonucleotide table that sets forth the sequences of oligonucleotides used in the Examples of the present specification.

INCORPORATION BY REFERENCE

All patents, publications, and patent applications cited in this specification are herein incorporated by reference as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A and FIG. 1B present illustrative examples of dual guide Type II CRISPR-Cas9 associated RNAs. FIG. 1A shows a two-RNA component Type II CRISPR-Cas9 system comprising a crRNA ( FIG. 1A, 101 ) and a tracrRNA ( FIG. 1A, 102 ). FIG. 1B illustrates the formation of base-pair hydrogen bonds between the crRNA and the tracrRNA to form secondary structure (see U.S. Published Patent Application No. 2014-0068797, published 6 Mar. 2014; see also Jinek M., et al., âA programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,â Science, 2012; 337:816-21). The figure presents an overview of and nomenclature for secondary structural elements of the crRNA and tracrRNA of the Streptococcus pyogenes Cas9 including the following: a spacer element ( FIG. 1B, 103 ); a first stem element comprising a lower stem element ( FIG. 1B, 104 ), a bulge element comprising unpaired nucleotides ( FIG. 1B, 105 ), and an upper stem element ( FIG. 1B, 106 ); a nexus element ( FIG. 1B, 107 ); a second hairpin element comprising a second stem element ( FIG. 1B, 108 ); and a third hairpin element comprising a third stem element ( FIG. 1B, 109 ). The figures are not proportionally rendered nor are they to scale. The locations of indicators are approximate.

FIG. 2 shows another example of a CRISPR-Cas9 associated RNA. The figure illustrates a single guide RNA (sgRNA) wherein the crRNA is covalently joined to the tracrRNA and forms a RNA polynucleotide secondary structure through base-pair hydrogen bonding (see, e.g., U.S. Published Patent Application No. 2014-0068797, published 6 Mar. 2014). The figure presents an overview of and nomenclature for secondary structural elements of a sgRNA of the Streptococcus pyogenes Cas9 including the following: a spacer element ( FIG. 2, 201 ); a first stem element comprising a lower stem element ( FIG. 2, 202 ), a bulge element comprising unpaired nucleotides ( FIG. 2, 205 ), and an upper stem element ( FIG. 2, 203 ); a loop element ( FIG. 2, 204 ) comprising unpaired nucleotides; (a first hairpin element comprises the first stem element and the loop element); a nexus element ( FIG. 2, 206 ); a second hairpin element comprising a second stem element ( FIG. 2, 207 ); and a third hairpin element comprising a third stem element ( FIG. 2, 208 ). (See, e.g., FIGS. 1 and 3 of Briner, A. E., et al., âGuide RNA Functional Modules Direct Cas9 Activity and Orthogonality,â Molecular Cell Volume 56, Issue 2, 23 Oct. 2014, Pages 333-339.) The figure is not proportionally rendered nor is it to scale. The locations of indicators are approximate.

FIG. 3A , FIG. 3B , FIG. 3C , FIG. 3D , FIG. 3E , FIG. 3F , FIG. 3G , and FIG. 3H present a variety of polynucleotides of the engineered Type II CRISPR-Cas9 systems of the present invention. The âsplit-nexus Cas9-associated polynucleotidesâ (sn-casPNs) of the present invention comprise two or more polynucleotides, wherein the polynucleotide backbone is broken within the nexus element. These figures present exemplary sn-casPN structures. Other modifications of sn-casPNs are described in the present specification. The figures are not proportionally rendered nor are they to scale. The indicators for locations corresponding to elements are only illustrative to provide reference points in the example polynucleotides.

Table 1 presents a series of indicators used consistently in FIG. 3A , FIG. 3B , FIG. 3C , FIG. 3D , FIG. 3E , FIG. 3F , FIG. 3G , and FIG. 3H .

TABLE 1

Numerical Indicators Used to Illustrate Regions of

Nucleotide Sequence Associated with Example sn-casPNs

Indicators and Corresponding Elements

Second Polynucleotide (sn2-casPN)

304 to 305 corresponds to a split nexus stem element nucleotide

sequence II

305 to 306 corresponds to a second connective nucleotide sequence

306 to 307 corresponds to a second stem element nucleotide sequence I

307 to 308 corresponds to a loop element nucleotide sequence

308 to 309 corresponds to a second stem element nucleotide sequence II

309 to 310 corresponds to a third connective nucleotide sequence

310 to 311 corresponds to a third stem element nucleotide sequence I

311 to 312 corresponds to a loop element nucleotide sequence

312 to 313 corresponds to a third stem element nucleotide sequence II

313 to 314 corresponds to a 3â² nucleotide sequence

First Polynucleotide (sn1-casPN)

315 to 316 corresponds to a split nexus stem element nucleotide

sequence I

316 to 317 corresponds to a first connective nucleotide sequence

317 to 320 corresponds to a first stem element nucleotide sequence I

317 to 318 corresponds to a lower stem element nucleotide sequence I

318 to 319 corresponds to a bulge element nucleotide sequence I

319 to 320 corresponds to an upper stem element nucleotide sequence I

320 to 321 corresponds to a loop element nucleotide sequence

Additional Polynucleotides (sn3-casPN, sn4-casPN)

321 to 324 corresponds to a first stem element nucleotide sequence II

321 to 322 corresponds to an upper stem element nucleotide sequence II

322 to 323 corresponds to a bulge element nucleotide sequence II

323 to 324 corresponds to a lower stem element nucleotide sequence II

324 to 325 corresponds to a nucleic acid target binding sequence

(a spacer element)

FIG. 3A illustrates an example of a split-nexus Cas9-associated three polynucleotide system. FIG. 3A, 301 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3A, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3A, 303 illustrates a third polynucleotide (sn3-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn3-casPN first stem element nucleotide sequence II form a first stem element, the first stem element comprising sn1-casPN lower stem element nucleotide sequence I/sn3-casPN lower stem element nucleotide sequence II form a lower stem element, and sn1-casPN upper stem element nucleotide sequence I/sn3-casPN upper stem element nucleotide sequence II form an upper stem element.

FIG. 3B illustrates an example of a split-nexus Cas9-associated two polynucleotide system. FIG. 3B, 326 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3B, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn1-casPN first stem element nucleotide sequence II form a first stem element, the first stem element comprising sn1-casPN lower stem element nucleotide sequence I/sn1-casPN lower stem element nucleotide sequence II forming a lower stem element, and sn1-casPN upper stem element nucleotide sequence I/sn1-casPN upper stem element nucleotide sequence II forming an upper stem element.

FIG. 3C illustrates an example of a split-nexus Cas9-associated three polynucleotide system. FIG. 3C, 327 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3C, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3C, 328 illustrates a third polynucleotide (sn3-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn3-casPN first stem element nucleotide sequence II form a first stem element.

FIG. 3D illustrates an example of a split-nexus Cas9-associated two polynucleotide system. FIG. 3D, 329 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3D, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn1-casPN first stem element nucleotide sequence II form a first stem element.

FIG. 3E illustrates an example of a split-nexus Cas9-associated four polynucleotide system. FIG. 3E, 301 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3E, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3E, 330 illustrates a third polynucleotide (sn3-casPN). FIG. 3E, 331 illustrates a spacer polynucleotide (sn4-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn3-casPN first stem element nucleotide sequence II form a first stem element, the first stem element comprising sn1-casPN lower stem element nucleotide sequence I/sn3-casPN lower stem element nucleotide sequence II forming a lower stem element, and sn1-casPN upper stem element nucleotide sequence I/sn3-casPN upper stem element nucleotide sequence II forming an upper stem element.

FIG. 3F illustrates an example of a split-nexus Cas9-associated three polynucleotide system. FIG. 3F, 332 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3F, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3F, 331 illustrates a spacer polynucleotide (sn4-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn1-casPN first stem element nucleotide sequence II form a first stem element, the first stem element comprising sn1-casPN lower stem element nucleotide sequence I/sn1-casPN lower stem element nucleotide sequence II forming a lower stem element, and sn1-casPN upper stem element nucleotide sequence I/sn1-casPN upper stem element nucleotide sequence II forming an upper stem element.

FIG. 3G illustrates an example of a split-nexus Cas9-associated four polynucleotide system. FIG. 3G, 327 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3G, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3G, 333 illustrates a third polynucleotide (sn3-casPN). FIG. 3G, 331 illustrates a spacer polynucleotide (sn4-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn3-casPN first stem element nucleotide sequence II form a first stem element.

FIG. 3H illustrates an example of a split-nexus Cas9-associated three polynucleotide system. FIG. 3H, 334 illustrates a first polynucleotide (sn1-casPN) that comprises a first portion of the split nexus element. FIG. 3H, 302 illustrates a second polynucleotide (sn2-casPN) that comprises a second portion of the split nexus element. FIG. 3H, 331 illustrates a spacer polynucleotide (sn4-casPN) that comprises a spacer element. Examples of polynucleotide secondary structures that form through base-pair hydrogen bonding between indicated sequences include the following: sn1-casPN split nexus stem element nucleotide sequence I/sn2-casPN split nexus stem element nucleotide sequence II form a split nexus stem element; sn2-casPN second stem element nucleotide sequence I/sn2-casPN second stem element nucleotide sequence II form a second stem element; sn2-casPN third stem element nucleotide sequence I/sn2-casPN third stem element nucleotide sequence II form a third stem element; sn1-casPN first stem element nucleotide sequence I/sn1-casPN first stem element nucleotide sequence II form a first stem element.

FIG. 4A presents modifications of Polynucleotide 1 (sn1-casPN) and Polynucleotide 2 (sn2-casPN) described above in FIG. 3A to FIG. 3H . FIG. 4B presents examples of further modifications to polynucleotide 1 (sn1-casRNA; described above in FIG. 3A to FIG. 3H ) and polynucleotide 3 (sn3-casRNA; described above in FIG. 3A , FIG. C, FIG. E, and FIG. 3G ) described above in FIG. FIG. 4A and FIG. 4B present examples of sn1-casPN, sn2-casPN, and sn3-casPN structures. Other modifications of sn1-casPN, sn2-casPN, and sn3-casPN are described in the present specification. The figures are not proportionally rendered nor are they to scale. The indicators for locations corresponding to elements are only illustrative to provide reference points in the example polynucleotides. Table 2 presents a series of indicators used consistently in FIG. 4A and FIG. 4B .

TABLE 2

Numerical Indicators Used to Illustrate Regions of Nucleotide

Sequences Associated with Examples of sn1-casPNs, sn2-casPNs,

and sn3-casPNs

Indicators and Corresponding Elements

Second Polynucleotide (sn2-casPN; second auxiliary polynucleotide;

first adjunct polynucleotide; second adjunct polynucleotide)

405 to 406 corresponds to a split nexus stem element nucleotide

sequence II

406 to 407 corresponds to a second connective nucleotide sequence

407 to 408 corresponds to a second stem element nucleotide sequence I

408 to 409 corresponds to a loop element nucleotide sequence

409 to 410 corresponds to a second stem element nucleotide sequence II

410 to 411 corresponds to a third connective nucleotide sequence

411 to 412 corresponds to a third stem element nucleotide sequence I

412 to 413 corresponds to a loop element nucleotide sequence

413 to 414 corresponds to a third stem element nucleotide sequence II

414 to 415 corresponds to a 3â² nucleotide sequence

405 to 418 corresponds to a second auxiliary polynucleotide

405 to 416 corresponds to a linker element nucleotide sequence II

416 to 417 corresponds to an affinity nucleotide sequence II

417 to 418 corresponds to an effector binding element nucleotide

sequence II

First Polynucleotide (sn1-casPN; auxiliary polynucleotide)

419 to 420 corresponds to a split nexus stem element nucleotide

sequence I

420 to 421 corresponds to a first connective nucleotide sequence

419 to 424 corresponds to a first auxiliary polynucleotide

419 to 422 corresponds to a linker element nucleotide sequence I

422 to 423 corresponds to an affinity nucleotide sequence I

423 to 424 corresponds to an effector binding element nucleotide

sequence I

421 to 425 corresponds to a lower stem element nucleotide sequence I

425 to 426 corresponds to a bulge element nucleotide sequence I

426 to 427 corresponds to an upper stem element nucleotide sequence I

427 to 428 corresponds to a first accessory polynucleotide

Third Polynucleotide (sn3-casPN; accessory polynucleotide)

429 to 430 corresponds to a second ac

CLAIMS

Claims ( 20 )

The invention claimed is:

1. One or more expression cassettes comprising:

one or more regulatory sequences operably linked to one or more polynucleotides encoding,

a first Type II CRISPR-Cas9-associated split-nexus polynucleotide having a 5â² end and a 3â² end (sn1-casPN) comprising, in the 5â² to 3â² direction, a first stem element nucleotide sequence I and a nexus stem element nucleotide sequence I,

a second Type II CRISPR-Cas9-associated split-nexus polynucleotide having a 5â² end and a 3â² end (sn2-casPN) comprising, in the 5â² to 3â² direction, a nexus stem element nucleotide sequence II and a second stem element nucleotide sequence I, wherein the nexus stem element nucleotide sequence I of the sn1-casPN and the nexus stem element nucleotide sequence II of the sn2-casPN are capable of forming a nexus stem element by base-pair hydrogen bonding between the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II,

a third Type II CRISPR-Cas9-associated polynucleotide having a 5â² end and a 3â² end (sn3-casPN) comprising, in the 5â² to 3â² direction, a DNA target binding sequence and a first stem element nucleotide sequence II, wherein the first stem element nucleotide sequence I of the sn1-casPN and the first stem element nucleotide sequence II of the sn3-casPN are capable of forming a first stem element by base-pair hydrogen bonding between the first stem element nucleotide sequence I and the first stem element nucleotide sequence II,

a first adjunct polynucleotide having a 5â² end and a 3â² end comprising a second stem element nucleotide sequence II, wherein the second stem element nucleotide sequence I of the sn2-casPN and the second stem element nucleotide sequence II of the first adjunct polynucleotide are capable of forming a second stem element by base-pair hydrogen bonding between the second stem element nucleotide sequence I and the second stem element nucleotide sequence II,

wherein the one or more regulatory elements are capable of facilitating expression of a Type II CRISPR-Cas9-associated polynucleotide composition comprising the sn1-casPN, the sn2-casPN, the sn3-casPN, and the first adjunct polynucleotide.

2. The one or more expression cassettes of claim 1 , wherein the nexus stem element nucleotide sequence I comprises a first auxiliary polynucleotide 3â² of the nexus stem element nucleotide sequence I.

3. The one or more expression cassettes of claim 1 , wherein the nexus stem element nucleotide sequence II comprises a second auxiliary polynucleotide 5â² of the nexus stem element nucleotide sequence II.

4. The one or more expression cassettes of claim 1 , wherein the nexus stem element nucleotide sequence I comprises a first auxiliary polynucleotide 3â² of the nexus stem element nucleotide sequence I and the nexus stem element nucleotide sequence II comprises a second auxiliary polynucleotide 5â² of the nexus stem element nucleotide sequence II.

5. The one or more expression cassettes of claim 4 , wherein

the first auxiliary polynucleotide further comprises an effector binding element nucleotide sequence I;

the second auxiliary polynucleotide further comprises an effector binding element nucleotide sequence II; and

the effector binding element nucleotide sequence I of the first auxiliary polynucleotide and the effector binding element nucleotide sequence II of the second auxiliary polynucleotide are capable of forming an effector binding element by base-pair hydrogen bonding between the effector binding element nucleotide sequence I and the effector binding element nucleotide sequence II.

6. The one or more expression cassettes of claim 4 , wherein

the first auxiliary polynucleotide further comprises, in the 5â² to 3â² direction, a linker element nucleotide sequence I and the effector binding element nucleotide sequence I;

the second auxiliary polynucleotide further comprises, in the 5â² to 3â² direction, the effector binding element nucleotide sequence II and a linker element nucleotide sequence II; and

the linker element nucleotide sequence I of the first auxiliary polynucleotide and the linker element nucleotide sequence II of the second auxiliary polynucleotide are capable of forming a linker element by base-pair hydrogen bonding between the linker element nucleotide sequence I and the linker element nucleotide sequence II.

7. The one or more expression cassettes of claim 4 , wherein the first auxiliary polynucleotide further comprises a hairpin and the second auxiliary polynucleotide further comprises a hairpin.

8. The one or more expression cassettes of claim 1 , wherein

the first adjunct polynucleotide further comprises, in the 5â² to 3â² direction, a loop element nucleotide sequence and the second stem element nucleotide sequence II; and

the 5â² end of the first adjunct polynucleotide is covalently bonded to the 3â² end of the sn2-casPN.

9. The one or more expression cassettes of claim 8 , wherein

the first stem element nucleotide sequence I further comprises, in the 5â² to 3â² direction, a loop element nucleotide sequence and the first stem element nucleotide sequence I; and

the 5â² end of the first stem element nucleotide sequence I is covalently bonded to the 3â² end of the first stem element nucleotide sequence II.

10. The one or more expression cassettes of claim 9 , wherein the one or more polynucleotides encoding further comprise,

a second adjunct polynucleotide having a 5â² end and a 3â² end comprising third stem element nucleotide sequence II; wherein the first adjunct polynucleotide comprises, in the 5â² to 3â² direction, the loop element nucleotide sequence, the second stem element nucleotide sequence II, and a third stem element nucleotide sequence I; and wherein the third stem element nucleotide sequence I of the first adjunct polynucleotide and the third stem element nucleotide sequence II of the second adjunct polynucleotide are capable of forming a third stem element by base-pair hydrogen bonding between the third stem element nucleotide sequence I and third stem element nucleotide sequence II.

11. The one or more expression cassettes of claim 10 , wherein

the second adjunct polynucleotide further comprises, in the 5â² to 3â² direction, a loop element nucleotide sequence and the third stem element nucleotide sequence II; and

5â² end of the second adjunct polynucleotide is covalently bonded to the 3â² end of the first adjunct polynucleotide.

12. The one or more expression cassettes of claim 1 , wherein

the first stem element nucleotide sequence I of the sn1-casPN further comprises, in the 5â² to 3â² direction, an upper stem element nucleotide sequence I, a bulge element nucleotide sequence I, and a lower stem element nucleotide sequence I;

the first stem element nucleotide sequence II of the sn3-casPN further comprises, in the 5â² to 3â² direction, a lower stem element nucleotide sequence II, a bulge element nucleotide sequence II, and an upper stem element nucleotide sequence II; and

the upper stem element nucleotide sequence I of the sn1-casPN and the upper stem element nucleotide sequence II of the sn3-casPN are capable of forming an upper stem element by base-pair hydrogen bonding between the upper stem element nucleotide sequence I and the upper stem element nucleotide sequence II and the lower stem element nucleotide sequence I of the sn1-casPN and the lower stem element nucleotide sequence II of the sn3-casPN are capable of forming a lower stem element by base-pair hydrogen bonding between the lower stem element nucleotide sequence I and the lower stem element nucleotide sequence II.

13. The one or more expression cassettes of claim 1 , further comprising an expression cassette comprising one or more regulatory sequences operably linked to a polynucleotide encoding a Cas9 protein.

14. The one or more expression cassettes of claim 13 , wherein the Cas9 protein comprises an inactive RuvC domain and an inactive HNH domain.

15. The one or more expression cassettes of claim 13 , wherein the Cas9 protein comprises an active RuvC domain, an active HNH domain, or an active RuvC domain and an active HNH domain.

16. The one or more expression cassettes of 13 , wherein the one or more regulatory sequences facilitate expression of the Type II CRISPR-Cas9-associated polynucleotide composition and the Cas9 protein in a eukaryotic cell.

17. The one or more expression cassettes of 1 , wherein the one or more regulatory sequences facilitate expression of the Type II CRISPR-Cas9-associated polynucleotide composition in a eukaryotic cell.

18. One or more vectors comprising:

the one or more expression cassettes of claim 1 .

19. The one or more vectors of claim 18 , wherein the one or more vectors are derived from a mammalian virus.

20. The one or more vectors of claim 19 , wherein the mammalian virus is at least one mammalian virus selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a retrovirus vector, and a lentivirus vector.

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