Patent Application
20230416710
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 17, 2022, is named 55921-717_301_SL.txt and is 1,351,136 bytes in size.
Cas enzymes along with their associated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (RNAs) appear to be a pervasive (˜45% of bacteria, ˜84% of archaea) component of prokaryotic immune systems, serving to protect such microorganisms against non-self nucleic acids, such as infectious viruses and plasmids by CRISPR-RNA guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse, containing a wide variety of nucleic acid-interacting domains. While CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage ability of CRISPR/Cas complexes has only been recognized relatively recently, leading to the use of recombinant CRISPR/Cas systems in diverse DNA manipulation and gene editing applications.
In some aspects, the present disclosure provides for a fusion endonuclease comprising: (a) an N-terminal sequence comprising at least part of a RuvC domain, a REC domain, or an HNH domain of an endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 696 or a variant thereof; and (b) a C-terminal sequence comprising WED, TOPO, or CTD domains of an endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 697-721 or variants thereof, wherein said N-terminal sequence and said C-terminal sequence do not naturally occur together in a same reading frame. In some embodiments, the endonuclease is a Class II, type II Cas endonuclease. In some embodiments, the endonuclease is a Class II, type V Cas endonuclease. In some embodiments, said N-terminal sequence and said C-terminal sequence are derived from different organisms. In some embodiments, said N-terminal sequence further comprises RuvC-I, BH, or RuvC-II domains. In some embodiments, said C-terminal sequence further comprises a PAM-interacting domain. In some embodiments, said fusion endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-27 or 108. In some embodiments, said fusion endonuclease is configured to bind to a PAM that is not nnRGGnT (SEQ ID NO: 53). In some embodiments, said fusion endonuclease is configured to bind to a PAM that comprises any one of SEQ ID NOs:46-52 or 54-66.
In some aspects, the present disclosure provides for an endonuclease comprising an engineered amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-27 or 108, or a variant thereof.
In some aspects, the present disclosure provides for an endonuclease comprising an engineered amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 109-110, or a variant thereof.
In some aspects, the present disclosure provides for a nucleic acid comprising a sequence encoding any of the endonucleases, fusion endonucleases, or Cas enzymes described herein. In some aspects, the sequence is codon-optimized for expression in a host cell. In some embodiments, the host cell is prokaryotic, eukaryotic, mammal, or human.
In some aspects, the present disclosure provides for a vector comprising any of the nucleic acid sequences described herein.
In some aspects, the present disclosure provides for a host cell comprising any of the vectors, systems, or nucleic acids described herein. In some embodiments, the host cell is prokaryotic, eukaryotic, mammal, or human.
In some aspects, the present disclosure provides for an engineered nuclease system, comprising: (a) any of the nucleases, Cas enzymes, or fusion endonucleases described herein; and (b) an engineered guide ribonucleic structure configured to form a complex with said endonuclease comprising: a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence; wherein said guide ribonucleic acid sequence is configured to bind to said endonuclease. In some embodiments, said guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind said endonuclease. In some embodiments, said endonuclease is derived from an uncultivated microorganism. In some embodiments, said endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, said endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, said system further comprises a source of Mg′. In some embodiments, said endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 8-12, 26-27, or 108, or a variant thereof. In some embodiments, said guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to non-degenerate nucleotides of any one of SEQ ID NOs: 33, 34, 44, 45, 78, 84, or 87.
In some aspects, the present disclosure provides for an engineered nuclease comprising: (a) a class II, type II Cas enzyme RuvC or HNH domain having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a RuvC or HNH domain of any one of SEQ ID NOs: 1-27, 108, or 109-110, or variants thereof and (b) a class II, type II Cas enzyme PAM-interacting (PI) domain having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a PAM-interacting (PI) domain any one of SEQ ID NOs: 1-27, 108, or 109-110, or variants thereof. In some embodiments, (a) and (b) do not naturally occur together. In some embodiments, said class II, type II Cas enzyme is derived from an uncultivated microorganism. In some embodiments, said endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, said engineered nuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-27 or a variant thereof.
In some aspects, the present disclosure provides for an engineered nuclease system, comprising: (a) any of the endonucleases described herein; and (b) an engineered guide ribonucleic structure configured to form a complex with said endonuclease comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and configured to bind to said endonuclease. In some embodiments, said guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind said endonuclease. In some embodiments, said guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 28-32 or 33-44, or a variant thereof. In some embodiments, the system further comprises a PAM sequence compatible with said nuclease adjacent to said target nucleic acid site. In some embodiments, said PAM sequence is located 3′ of said target deoxyribonucleic acid sequence. In some embodiments, said PAM sequence is located 5′ of said target deoxyribonucleic acid sequence. In some embodiments, said PAM sequence comprises any one of SEQ ID NOs:46-66.
In some aspects, the present disclosure provides for a method of targeting the albumin gene, comprising introducing any of the systems described herein to a cell, wherein said guide ribonucleic acid sequence is configured to hybridize to a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity any one of SEQ ID NOs: 67-86. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of targeting the HAO1 gene or locus, comprising introducing any of the systems described herein to a cell, wherein said guide ribonucleic acid sequence is configured to hybridize to a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 611-633. In some embodiments, said guide ribonucleic acid sequence is configured to hybridize to a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 615, 618, 620, 624, or 626. In some embodiments, said guide ribonucleic acid comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:645-684. In some embodiments, said guide ribonucleic acid comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 645-649, 652-656, 660-671, 674-675, or 681-684, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a targeting sequence of any one of SEQ ID NOs: 645-649, 652-656, 660-671, 674-675, or 681-684. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some embodiments, the present disclosure provides for a method of disrupting an HAO-1 locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said HAO-1 locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 611-626 or 627-633. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 618, 620, 624, or 626, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a targeting sequence of any one of SEQ ID NOs: 618, 620, 624, or 626. In some embodiments, said engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9 or Table 12. In some embodiments, the cell is a mammalian cell. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of disrupting a TRAC locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said TRAC locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 139-158; or wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 119-138. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises the fusion endonuclease having at least 55% identity to SEQ ID NO:10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 121, 132, 136, 130, 134, 135, or 137, or a sequence having at least 80% identity to a targeting sequence of any one of SEQ ID NOs: 121, 132, 136, 130, 134, 135, or 137. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7A. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some embodiments, the present disclosure provides for a method of disrupting a B2M locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said B2M locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 185-210; or wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 159, 165, 168, 174, or 184, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a targeting sequence of any one of SEQ ID NOs: 159, 165, 168, 174, or 184. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7B. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of disrupting a TRBC1 locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said TRBC1 locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 252-292; or wherein the engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA is comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 211, 212, 215, 241, or 242, or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a targeting sequence of any one of SEQ ID NOs: 211, 212, 215, 241, or 242. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7C. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of disrupting a TRBC2 locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said TRBC2 locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 338-382; or wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the class 2, type II Cas endonuclease any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 296, 306, or 332, or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a targeting sequence of any one of SEQ ID Nos: 296, 306, or 332. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7C. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of disrupting an ANGPTL3 locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said ANGPTL3 locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80% identity to SEQ ID NOs: 478-572; or wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 383-477. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 419, 425, 431, 439, 447, 453, 461, 467, 471, or 473, or a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 419, 425, 431, 439, 447, 453, 461, 467, 471, or 473. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7D. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for a method of disrupting a PCSK9 locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said PCSK9 locus, wherein said engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs: 588-602; or wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 573-587. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease comprising a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 574, 578, 581, or 585. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 7E. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some embodiments, the present disclosure provides for a method of disrupting an albumin locus in a cell, comprising introducing to said cell: (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a targeting sequence configured to hybridize to a region of said albumin locus, wherein said engineered guide RNA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 67-86 or 646-695, or wherein said engineered guide RNA comprises a targeting sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a targeting sequence of any one of SEQ ID NOs: 67-86 or 646-695. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease. In some embodiments, said class 2, type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises any of the type II Cas endonucleases described herein. In some embodiments, said class 2, type II Cas endonuclease comprises a fusion endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, said engineered guide RNA comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, said engineered guide RNA is complementary to or comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 67, 68, 70, 71, 72, 76, 79, 80, 647, 648, 649, 653, 654, 655, 656, 673, 680, 681, or 682. In some embodiments, said engineered guide RNA comprises a nucleotide sequence of any one of the guide RNAs from Table 6. In some embodiments, introducing to said cell further comprises contacting said cell with a nucleic acid or vector encoding said fusion protein or said guide polynucleotide. or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said vector or nucleic acid. In some embodiments, introducing to said cell further comprises contacting said cell with a ribonucleoprotein complex (RNP) comprising said fusion protein or said guide polynucleotide or comprises contacting said cell with a lipid nanoparticle (LNP) comprising said RNP.
In some aspects, the present disclosure provides for an endonuclease comprising an engineered amino acid sequence having at least 55% sequence identity to any one of SEQ ID NOs: 1-27, 108, or 109-110.
In some aspects, the present disclosure provides an engineered nuclease system, comprising the endonuclease described herein, and an engineered guide ribonucleic structure configured to form a complex with the endonuclease comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and a tracr ribonucleic acid sequence configured to bind to said endonuclease. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the system further comprises a source of MG′.
In some aspects, the present disclosure provides for an engineered nuclease comprising: (a) a class II, type II Cas enzyme RuvC and HNH domain having at least 55% sequence identity to a RuvC and HNH domain of any one of SEQ ID NOs: 1-27, 108, or 109-110; and (b) a class II, type II Cas enzyme PAM-interacting (PI) domain having at least 55% sequence identity to a PAM-interacting (PI) domain any one of SEQ ID NOs: 1-27, 108, or 109-110. In some embodiments, (a) and (b) do not naturally occur together. In some embodiments, the class II, type II Cas enzyme is derived from an uncultivated microorganism. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the engineered nuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs: 1-27.
In some aspects, the present disclosure provides for an engineered nuclease system, comprising: an endonuclease according to any of the aspects or embodiments described herein; and an engineered guide ribonucleic structure configured to form a complex with the endonuclease comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 28-32 or 33-44, or a variant thereof. In some embodiments, the system further comprises a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located 3′ of the target deoxyribonucleic acid sequence. In some embodiments, the PAM sequence comprises any one of SEQ ID NOs:46-66.
In some embodiments, the present disclosure provides for an engineered single-molecule heterologous guide polynucleotide compatible with a class II, type II enzyme according to any of the aspects or embodiments described herein, wherein the heterologous guide polynucleotide comprises chemical modifications according to any one of SEQ ID NOs: 645-684.
In some aspects, the present disclosure provides for a method of targeting the albumin gene, comprising introducing a system according to any one of the aspects or embodiments described herein to a cell, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence comprising any one of SEQ ID NOs: 67-86.
In some aspects, the present disclosure provides for a method of targeting the HAO1 gene, comprising introducing a system according to any one of the aspects or embodiments described herein to a cell, wherein the guide ribonucleic acid sequence is configured to hybridize to any one of SEQ ID NOs: 611-633. In some embodiments, the guide ribonucleic acid sequence is configured to hybridize to any one of SEQ ID NOs: 615, 618, 620, 624, or 626. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOs:645-684. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOs: 645-649, 652-656, 660-671, 674-675, or 681-684.
In some aspects, the present disclosure provides cells comprising the endonucleases described herein. In some aspects, the present disclosure provides cells comprising any nucleic acid molecule described herein. In some aspects, the present disclosure provides cells comprising any engineered nuclease system described herein.
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
The Sequence Listing filed herewith provides example polynucleotide and polypeptide sequences for use in methods, compositions, and systems according to the disclosure. Below are example descriptions of sequences therein.
SEQ ID NOs: 1-27 show the full-length peptide sequences of MG3-6 chimeric nucleases.
SEQ ID NO: 108 shows the nucleotide sequence of an MG3-6/3-4 nuclease containing 5′ UTR, NLS, CDS, NLS, 3′ UTR, and polyA tail.
SEQ ID NOs: 28-45 and 605-610 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6 chimeric nuclease.
SEQ ID NOs: 46-59 show the natural PAM specificities of various effectors.
SEQ ID NOs: 60-66 show the PAM specificities of chimeric nucleases described herein.
SEQ ID NO: 603 shows the DNA coding sequence for MG3-6/3-4.
SEQ ID NO: 604 shows the protein sequence of the MG3-6/3-4 cassette coding sequence.
SEQ ID NOs: 109-110 show the full-length peptide sequences of MG29-1 chimeric nucleases.
SEQ ID NOs: 111-113 show the nucleotide sequences of sgRNAs engineered to function with an MG29-1 chimeric nuclease.
SEQ ID NOs: 114-116 show the natural PAM specificities of various effectors.
SEQ ID NO: 117 shows the PAM specificity of a chimeric nuclease described herein.
SEQ ID NOs: 119-138 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRAC.
SEQ ID NOs: 139-158 show the DNA sequences of TRAC target sites.
SEQ ID NOs: 159-184 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target B2M.
SEQ ID NOs: 185-210 show the DNA sequences of B2M target sites.
SEQ ID NOs: 211-251 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRBC1.
SEQ ID NOs: 252-292 show the DNA sequences of TRBC1 target sites.
SEQ ID NOs: 293-337 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target TRBC2.
SEQ ID NOs: 338-382 show the DNA sequences of TRBC2 target sites.
SEQ ID NOs: 383-477 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target ANGPTL3.
SEQ ID NOs: 478-572 show the DNA sequences of ANGPTL3 target sites.
SEQ ID NOs: 573-587 show the nucleotide sequences of sgRNAs engineered to function with an MG3-6/3-4 nuclease in order to target PCSK9.
SEQ ID NOs: 588-602 show the DNA sequences of PCSK9 target sites.
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)) (which is entirely incorporated by reference herein).
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.
As used herein, a “cell” generally refers to a biological cell. A cell may be the basic structural, functional, or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).
The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide may comprise a synthetic nucleotide. A nucleotide may comprise a synthetic nucleotide analog. Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores). Labeling may also be carried out with quantum dots. Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).
The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function. A polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.
The terms “transfection” or “transfected” generally refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to generally refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids may include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues may refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.
As used herein, the “non-native” can generally refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein. Non-native may refer to affinity tags. Non-native may refer to fusions. Non-native may refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions, or deletions. A non-native sequence may exhibit or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) that may also be exhibited by the nucleic acid or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence may be linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.
The term “promoter”, as used herein, generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. A ‘basal promoter’, also referred to as a ‘core promoter’, may generally refer to a promoter that contains all the basic elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters comprise, in some instances, a TATA-box or a CAAT box.
The term “expression”, as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which may comprise promoter or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.
As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.
A “functional fragment” of a DNA or protein sequence generally refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence. A biological activity of a DNA sequence may be its ability to influence expression in a manner attributed to the full-length sequence.
As used herein, an “engineered” object generally indicates that the object has been modified by human intervention. According to non-limiting examples: a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property. An “engineered” system comprises at least one engineered component.
As used herein, “synthetic” and “artificial” are used interchangeably to refer to a protein or a domain thereof that has low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains.
The term “tracrRNA” or “tracr sequence”, as used herein, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity or sequence similarity to a wild type example tracrRNA sequence (e.g., a tracrRNA from S. pyogenes S. aureus, etc. or SEQ ID NOs: *_*). tracrRNA can refer to a nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity or sequence similarity to a wild type example tracrRNA sequence (e.g., a tracrRNA from S. pyogenes S. aureus, etc). tracrRNA may refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. A tracrRNA may refer to a nucleic acid that can be at least about 60% identical to a wild type example tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild type example tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.
As used herein, a “guide nucleic acid” can generally refer to a nucleic acid that may hybridize to another nucleic acid. A guide nucleic acid may be RNA. A guide nucleic acid may be DNA. The guide nucleic acid may be programmed to bind to a sequence of nucleic acid site-specifically. The nucleic acid to be targeted, or the target nucleic acid, may comprise nucleotides. The guide nucleic acid may comprise nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid may be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore may not be complementary to the guide nucleic acid may be called noncomplementary strand. A guide nucleic acid may comprise a polynucleotide chain and can be called a “single guide nucleic acid.” A guide nucleic acid may comprise two polynucleotide chains and may be called a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” may be inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment that can be referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence.” A nucleic acid-targeting segment may comprise a sub-segment that may be referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment”.
The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov); CLUSTALW with parameters of; the Smith-Waterman homology search algorithm with parameters of a match of 2, a mismatch of −1, and a gap of −1; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.
As used herein, the term “RuvC_III domain” generally refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC_I, RuvC_II, and RuvC_III). A RuvC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMIs) built based on documented domain sequences (e.g., Pfam HMM PF18541 for RuvC_III).
As used herein, the term “Wedge” (WED) domain generally refers to a domain (e.g. present in a Cas protein) interacting primarily with repeat:anti-repeat duplex of the sgRNA and PAM duplex. A WED domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences.
As used herein, the term “PAM interacting domain” or “PI domain” generally refers to a domain interacting with the protospacer-adjacent motif (PAM) external to the seed sequence in a region targeted by a Cas protein. Examples of PAM-interacting domains include, but are not limited to, Topoisomerase-homology (TOPO) domains and C-terminal domains (CTD) present in Cas proteins. A PAM interacting domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences.
As used herein, the term “REC domain” generally refers to a domain (e.g. present in a Cas protein) comprising at least one of two segments (REC1 or REC2) that are alpha helical domains thought to contact the guide RNA. A REC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMIs) built based on documented domain sequences (e.g., Pfam PF19501 for domain REC1).
As used herein, the term “BH domain” generally refers to a domain (e.g. present in a Cas protein) that is a bridge helix between NUC and REC lobes of a Type II Cas enzyme. A BH domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam PF16593 for domain BH).
As used herein, the term “HNH domain” generally refers to an endonuclease domain having characteristic histidine and asparagine residues. An HNH domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMIs) built based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).
Included in the current disclosure are variants of any of the enzymes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g. non-conserved residues without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity any one of the systems described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease are not disrupted. In some embodiments, a functional variant of any of the systems described herein lack substitution of at least one of the conserved or functional residues described herein. In some embodiments, a functional variant of any of the systems described herein lacks substitution of all of the conserved or functional residues described herein.
Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for example, Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd Edition (December 1993))). The following eight groups each contain amino acids that are conservative substitutions for one another:
Overview
The discovery of new Cas enzymes with unique functionality and structure may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR/Cas enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR/Cas systems documented and speed the discovery of new oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX/CasY CRISPR systems from metagenomic analysis of natural microbial communities.
CRISPR/Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, CRISPR/Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40 bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins/enzymes. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.
Class I CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.
Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cash) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nucleases function primarily as DNA nucleases.
Type III CRISPR systems may be characterized by the presence of a central nuclease, known as Cas10, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre-crRNA using a Cash-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).
Type IV CRISPR-Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.
Class II CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.
Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g. Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are documented as DNA nucleases. Type 2 effectors generally exhibit a structure consisting of a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.
Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g. Cas12) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again documented as DNA nucleases. Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., Cas12a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.
Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (e.g. Cas13) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to, in some embodiments, not require a tracrRNA for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.
Because of their simpler architecture, Class II CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease/genome editing applications.
One of the early adaptations of such a system for in vitro use can be found in Jinek et al. (Science. 2012 Aug. 17; 337(6096):816-21, which is entirely incorporated herein by reference). The Jinek study first described a system that involved (i) recombinantly-expressed, purified full-length Cas9 (e.g., a Class II, Type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature ˜42 nt crRNA bearing a ˜20 nt 5′ sequence complementary to the target DNA sequence to be cleaved followed by a 3′ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg2+. Jinek later described an improved, engineered system wherein the crRNA of (ii) is joined to the 5′ end of (iii) by a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.
Mali et al. (Science. 2013 Feb. 15; 339(6121): 823-826), which is entirely incorporated herein by reference, later adapted this system for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g., a Class II, Type II Cas enzyme) under a suitable mammalian promoter with a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (having a 5′ sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence joined to a 3′ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter).
Engineered Nucleases
In some aspects, the present disclosure relates to the engineering of novel nucleic acid-guided nucleases and systems. In some embodiments, the engineered nucleases are functional in prokaryotic or eukaryotic cells for in vitro, in vivo or ex vivo applications. In some embodiments, the present disclosure relates to the engineering and optimization of systems, methods and compositions used for genome engineering involving sequence targeting, such as genome perturbation or gene-editing, that relate to nucleic acid-guided nuclease systems and components thereof.
In some aspects, the present disclosure provides engineered nucleases which may include nucleic acid guided nucleases, chimeric nucleases, and nuclease fusions.
Chimeric or Fusion Engineered Nucleases
Chimeric engineered nucleases as described herein may comprise one or more fragments or domains, and the fragments or domains may be of a nuclease, such as nucleic acid-guided nuclease, orthologs of organisms of genus, species, or other phylogenetic groups described herein. The fragments may be from nuclease orthologs of different species. A chimeric engineered nuclease may be comprised of fragments or domains from at least two different nucleases. A chimeric engineered nuclease may be comprised of fragments or domains from nucleases from at least two different species. A chimeric engineered nuclease may be comprised of fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleases or nucleases from different species. In some embodiments, a chimeric engineered nuclease comprises more than one fragment or domain from one nuclease, wherein the more than one fragment or domain are separated by fragments or domains from a second nuclease. In some examples, a chimeric engineered nuclease comprises 2 fragments, each from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 3 fragments, each from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 4 fragments, each from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 5 fragments, each from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 3 fragments, wherein at least one fragment is from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 4 fragments, wherein at least one fragment is from a different protein or nuclease. In some examples, a chimeric engineered nuclease comprises 5 fragments, wherein at least one fragment is from a different protein or nuclease.
Junctions between fragments or domains from different nucleases or species can occur in stretches of unstructured regions. Unstructured regions may include regions which are exposed within a protein structure or are not conserved within various nuclease orthologs.
MG Chimeric Enzymes
The CRISPR effectors described herein have natural PAM specificities (see
In some aspects, the present disclosure provides for a fusion endonuclease comprising: (a) an N-terminal sequence comprising RuvC, REC, or HNH domains of a Cas endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 696 or a variant thereof; and (b) a C-terminal sequence comprising WED, TOPO, or CTD domains of a Cas endonuclease having at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 697-721 or variants thereof. In some embodiments the fusion endonuclease comprises RuvC, REC, and HNH domains in (a). In some embodiments, the fusion endonuclease comprises RuvC and HNH domains in (a). In some embodiments, the fusion endonuclease comprises WED, TOPO, and CTD domains in (b). In some embodiments, the N-terminal sequence and the C-terminal sequence do not naturally occur together in a same reading frame. In some embodiments, the N-terminal sequence and the C-terminal sequence are derived from different organisms. In some embodiments, the N-terminal sequence further comprises RuvC-I, BH, and RuvC-II domains. In some embodiments, the C-terminal sequence further comprises a PAM-interacting domain. In some embodiments, the fusion Cas endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity sequence identity to any one of SEQ ID NOs: 1-27 or 108. In some embodiments, the fusion endonuclease is configured to bind to a PAM that is not nnRGGnT (SEQ ID NO: 53). In some embodiments, the fusion endonuclease is configured to bind to a PAM that comprises any one of SEQ ID NOs:46-52 or 54-66.
In some aspects, the present disclosure provides an endonuclease comprising an engineered nucleic acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-27, 108, or 109-110. In one aspect, the present disclosure provides an endonuclease comprising an engineered nucleic acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 8-12, 26-27, or 108. In one aspect, the present disclosure provides an engineered nuclease system, comprising: the endonuclease described herein; and an engineered guide ribonucleic structure configured to form a complex with the endonuclease comprising: a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and configured to bind to the endonuclease. In some embodiments, and the engineered guide ribonucleic acid sequence further comprises a tracr ribonucleic acid sequence. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the system further comprises a source of Mg′.
In some aspects, the present disclosure provides for an engineered nuclease system comprising: (a) any of the endonucleases described herein (e.g. a fusion endonuclease comprising: (a) an N-terminal sequence comprising RuvC, REC, or HNH domains of a Cas endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 696 or a variant thereof; and (b) a C-terminal sequence comprising WED, TOPO, or CTD domains of a Cas endonuclease having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 697-721 or variants thereof; and (b) an engineered guide ribonucleic structure configured to form a complex with the endonuclease comprising: a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence; wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the system further comprises a source of Mg 2+. In some embodiments, the endonuclease comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 8-12, 26-27, or 108. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 33, 34, 44, 78, 84, or 87.
Systems of the present disclosure may be used for various applications, such as, for example, nucleic acid editing (e.g., gene editing), binding to a nucleic acid molecule (e.g., sequence-specific binding). Such systems may be used, for example, for addressing (e.g., removing or replacing) a genetically inherited mutation that may cause a disease in a subject, inactivating a gene in order to ascertain its function in a cell, as a diagnostic tool to detect disease-causing genetic elements (e.g. via cleavage of reverse-transcribed viral RNA or an amplified DNA sequence encoding a disease-causing mutation), as deactivated enzymes in combination with a probe to target and detect a specific nucleotide sequence (e.g. sequence encoding antibiotic resistance int bacteria), to render viruses inactive or incapable of infecting host cells by targeting viral genomes, to add genes or amend metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites, to establish a gene drive element for evolutionary selection, to detect cell perturbations by foreign small molecules and nucleotides as a biosensor.
Chimera sequences were codon optimized for E. coli expression via Integrated DNA Technologies (IDT) website, and synthesized and cloned into pET21 vector at Twist Bioscience unless otherwise specified. To construct pET21-MG3-6+MG15-1 (WP) and pET21-MG3-6+MG15-1 (P), gene fragments were amplified from pMGX3-6 and pMGX15-1 using primers P441-P446. The resulting PCR products were purified by Zymo Gel DNA Recovery Kit and assembled into pAL3 (digested by ClaI and XhoI) via NEBUilder HiFi DNA assembly. DNA sequences of cloned chimeric genes were confirmed by Sanger sequencing service offered by Elim Biopharm.
CRISPR Type II endonucleases utilized herein were predicted to have nuclease activity based on the presence of putative HNH and RuvC catalytic residues. In addition, structural predictions suggested residues involved in guide, target, and recognition of and interaction with a PAM. Based on the location of important residues, the predicted domain architecture of Type II CRISPR endonucleases comprised three RuvC domains, an HNH endonuclease domain, a recognition domain and PAM interacting domain, among others. For genomic sequences encoding a full-length Type II endonuclease next to a CRISPR array, we predicted tracrRNA sequences, which were engineered to be used by the nuclease as single guide RNAs.
A multiple sequence alignment of selected RNA guided CRISPR Type II endonuclease sequences were performed using the built-in MUSCLE aligner on Geneious Primer Software (available at https://www.geneious.com/prime) (see
The PAM sequences of nucleases utilized herein were determined via expression in either an E. coli lysate-based expression system or reconstituted in vitro translation (myTXTL, Arbor Biosciences or PURExpress, New England Biolabs). The E. coli codon optimized protein sequence was transcribed and translated from a PCR fragment under control of a T7 promoter. This mixture was diluted into a reaction buffer (10 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2) with protein-specific sgRNA and a PAM plasmid library (PAM library U67/U40). The library of plasmids contained a spacer sequence matching that in the single guide followed by 8N mixed bases, a subset of which were presumed to have the correct PAM. After 1-3 h, the reaction was stopped and the DNA was recovered via a DNA clean-up kit, e.g. Zymo DCC, AMPure XP beads, QiaQuick etc. The DNA was subjected to a blunt-end ligation reaction which added adapter sequences to cleaved library plasmids while leaving intact circular plasmids unchanged. A PCR was performed with primers (LA065 and LA125) specific to the library and the adapter sequence and resolved on a gel to identify active protein complexes (see
The single guide (sgRNA) structures used herein comprised a structure of: 5′ 22nt protospacer-repeat—tracr—3′. 20 single guides targeting mouse albumin intron 1 were designed using Geneious Prime Software (https://www.geneious.com/prime/). In some instances, guides were chemically synthesized by IDT and included a chemical modification of the guide that had been optimized by IDT to improve the performance of Cas9 guides (“Alt-R” modifications).
The coding sequences (CDS) encoding the chimeras (e.g. MG3-6+MG3-4 (SEQ ID NO: 10)) were codon-optimized for mouse and chemically synthesized at Twist biosciences. The CDS were cloned into mRNA production vector pMG010. The architecture of pMG010 comprised the sequence of elements: T7 promotor—5′UTR—start codon—nuclear localization signal 1—CDS—nuclear localization signal 2—stop codon—3′ UTR—107 nucleotide polyA tail (SEQ ID NO: 108). A plasmid pMG010 containing the MG3-6+MG 3-4 CDS was purified from a 200 ml bacterial culture using an EndoFree Plasmid Kit (Qiagen). The vector was digested with SapI overnight in order to linearize the plasmid downstream of the polyA tail. The linearized vector was purified using phenol/chloroform DNA extraction. In vitro transcription was carried out using HiT7 T7 RNA polymerase (New England Biolabs) at 50° C. for 1 h. In vitro transcribed mRNA was treated with DNase for 10 min at 37° C., and the mRNA was purified using the MEGAclear Transcription Clean-up kit (Thermo Fisher). mRNA was quantified by absorbance at 260 nm and its size and purity was assessed by automated electrophoresis (TapeStation, Agilent) and demonstrated to be of the expected size.
300 ng of mRNA and 350 ng of each single guide RNA (sgRNA) of SEQ ID NOs: 67-86 were co-transfected into Hepa1-6 cells as follows. One Day before transfection Hepa1-6 cells were seeded into 24 wells at a density to achieve 70% confluency 24 h later. The following day 25 μl of OptiMEM media and 1.25 μl of Lipofectamine Messenger Max Solution (Thermo Fisher) were mixed and vortexed for 5 s to make solution A. In a separate tube 300 ng of the MG3-6+MG3-4 chimera mRNA and 350 ng of a single guide were mixed together with 25 μl of OptiMEM to make Solution B. Solution A and B were mixed and incubated for 10 min at room temperature then added directly to the Hepa1-6 cells. Two days post transfection the media was aspirated, and genomic DNA was purified following the instructions from Purelink Genomic DNA mini kit (Thermo Fisher) (see
Primers flanking the regions of the genome targeted by the single guide RNAs (e.g. the albumin gene) were designed. PCR amplification using primers 57F (SEQ ID NO: 97) and 1072R (SEQ ID NO: 98) was performed using Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher) resulting in a PCR product of 1016 bp. PCR products were purified and concentrated using DNA clean & concentrator 5 (Zymo Research) and 100 ng of PCR product subjected to Sanger sequencing (ELIM Biosciences) using 8 pmoles of individual sequencing primers (132F, 282F, 446R, and 460F, SEQ ID NOs: 99-102). Sanger sequencing results were analyzed by using an algorithm called Inference of CRISPR edits (available at https://github.com/synthego-open/ice) and data was plotted using GradPrism (see
Guide RNA for the MG3-6/3-4 nuclease targeting exons 1 to 4 of the mouse HAO-1 gene (encodes glycolate oxidase) were identified in silico by searching for the PAM sequence 3′ NNAAA(A/T)N 5′. A total of 23 guides with the fewest predicted off-target sites in the mouse genome were chemically synthesized as single guide RNAs. 300 ng mRNA and 120 ng single guide RNA were transfected into Hepa1-6 cells as follows. One day prior to transfection, Hepa1-6 cells that have been cultured for less than 10 days in DMEM, 10% FBS, 1×NEAA media, without Pen/Strep, were seeded into a TC-treated 24 well plate. Cells were counted, and the equivalent volume to 60,000 viable cells were added to each well. Additional pre-equilibrated media was added to each well to bring the total volume to 500 μL. On the day of transfection, 254, of OptiMEM media and 1.25 ul of Lipofectamine Messenger Max Solution (Thermo Fisher) were mixed in a mastermix solution, vortexed, and allowed to sit for at least 5 minutes at room temperature. In separate tubes, 300 ng of the MG3-6-MG-3-4-encoding mRNA (SEQ ID NO: 108) and 120 ng of the sgRNA (scaffold sequence SEQ ID NO:34) were mixed together with 254, of OptiMEM media, and vortexed briefly. The appropriate volume of MessengerMax solution was added to each RNA solution, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were allowed to incubate for 10 minutes at room temperature, then added directly to the Hepa1-6 cells. Two days post transfection, the media was aspirated off of each well of Hepa1-6 cells and genomic DNA was purified by automated magnetic bead purification, via the KingFisher Flex with the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit. The activity of the guides is summarized in Tables 2 and 3, while the primers used are summarized in Table 4.
We designed 40 different chemically modified guides (named mAlb3634-34-0 to mAlb3634-34-44) and tested the activity of 39 of these guides. One guide, mH3634-34-32, failed RNA synthesis, thus it was not tested. The guide spacer sequence we chose as a model to insert various chemical modifications was mAlb3634-34 (targeting albumin intron 1) as it proved to be the most active guide in a guide screen in the mouse hepatocyte cell line Hepa1-6 cells (Table 5 and
The sgRNA of MG3-6/3-4 comprises a spacer located at the 5′ end followed by the CRISPR repeat and the trans-activating CRISPR RNA (tracr). The CRISPR repeat and the tracr are identical to that of the MG3-6 nuclease (
The editing activity of 39 single guides with the exact same base sequence but different chemical modifications was evaluated in Hepa1-6 cells by co-transfection of mRNA encoding MG3-6/3-4 and the guide; the results are shown in Table 6 and
A guide with the same base sequence and a commercially available chemical modification called AltR1/AltR2 was used as a control. The spacer sequence in these guides targets a 22-nucleotide region in albumin intron1 of the mouse genome. Guide mAlb3634-34-0 (no chemical modifications) showed 72% activity relative to the AltR1/AltR2 guide. Guide mAlb3634-34-1 showed 124% activity relative to the AltR1/AltR2 guide, showing the importance of stability of guides for editing: mAlb3634-34-1 is more stable than mAlb3634-34-0 (
In order to test the stability of these chemically modified guides compared to the guide with no chemical modification (native RNA), a stability assay using crude cell extracts was used. Crude cell extracts from mammalian cells were selected because they contain the mixture of nucleases that a guide RNA will be exposed to when delivered to mammalian cells in vitro or in vivo. Hepa1-6 cells were collected by adding 3 ml of cold PBS per 15 cm dish of confluent cells and releasing the cells from the surface of the dish using a cell scraper. The cells were pelleted at 200 g for 10 min and frozen at −80° C. for future use. For the stability assays, cells were resuspended in 4 volumes of cold PBS (e.g. for a 100 mg pellet, cells were resuspended in 400 ul of cold PBS). Triton X-100 was added to a concentration of 0.2% (v/v), cells were vortexed for 10 seconds, put on ice for 10 minutes, and vortexed again for 10 seconds. Triton X-100 is a mild non-ionic detergent that disrupts cell membranes but does not inactivate or denature proteins at the concentration used. Stability reactions were set up on ice and comprised 2011.1 of cell crude extract with 2 pmoles of each guide (1 ul of a 2 uM stock). Six reactions were set up per guide comprising: input, 0.5 hour, 1 hour, 4 hours, 9 hours, and in some cases 21 hours (The time in hours referring to the length of time each sample was incubated). Samples were incubated at 37° C. from 0.5 hours up to 21 hours while the input control was left on ice for 5 minutes. After each incubation period, the reaction was stopped by adding 300 ul of a mixture of phenol and guanidine thiocyanate (Tri reagent, Zymo Research), which immediately denatures all proteins and efficiently inhibits ribonucleases and facilitates the subsequent recovery of RNA. After adding Tri Reagent, the samples were vortexed for 15 seconds and stored at −20° C. RNA was extracted from the samples using Direct-zol RNA miniprep kit (Zymo Research) and eluted in 100 ul of nuclease-free water. Detection of the modified guide was performed using Taqman RT—qPCR using the Taqman miRNA Assay technology (Thermo Fisher), and primers and probes were designed to specifically detect the sequence in the mAlb3634-34 sgRNA, which is the same for all of the guides. Data was plotted as a function of percentage of sgRNA remaining in relation to the input sample (Tables 7 and 8;
The stability assays showed that introducing three 2′-O-methyls and three PS bonds in the 5′ and 3′ end of the guides significantly improved stability (
To expand the capability of rapid PAM exchange beyond type II nucleases, three type V-A nucleases were chosen for protein recombination. The breakpoint was chosen based on the predicted structural information (Table 1). Similar to type II enzyme recombinants, the type V chimera showed activity when proteins were recombined from a closely related family. In vitro PAM enrichment and NGS analysis revealed a consistent result that the PAM of a chimera is inherited from C-terminal parent. It may be possible to avoid potential structural disruptions of protein recombination from distantly related families by utilizing breakpoint optimization (
Nucleofection of MG3-6/4 RNPs (104 pmol protein/300 pmol guide) comprising sgRNAs described below in Table 7A and SEQ ID NOs: 119-158 was performed into HEK293T cells (200,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (
Nucleofection of MG3-6/4 RNPs (104 pmol protein/300 pmol guide) comprising sgRNAs described below in Table 7B and SEQ ID NOs: 159-210 was performed into HEK293T cells (200,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (
Primary T cells were purified from PMBCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of MG3-6/4 RNPs (104 pmol protein/120 pmol guide) comprising sgRNAs described in Table 7A and SEQ ID NOs: 119-158 was performed into T cells (200,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing. For analysis by flow cytometry, 3 days post-nucleofection, 100,000 T cells were stained with anti-CD3 antibody for 30 minutes at 4C and analyzed on an Attune Nxt flow cytometer (
Primary T cells were purified from PMBCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of MG3-6/4 RNPs (104 pmol protein/120 pmol guide) comprising sgRNAs described in Table 7B and SEQ ID NOs: 159-210 was performed into T cells (200,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (
Primary T cells were purified from PBMCs using a negative selection kit (Miltenyi) according to the manufacturer's recommendations. Nucleofection of MG3-6/4 RNPs (104 pmol protein/120 pmol guide) comprising sgRNAs described below in Table 7C below and SEQ ID NOs: 211-382 was performed into T cells (200,000) using the Lonza 4D electroporator. For analysis by flow cytometry, 3 days post-nucleofection, 100,000 T cells were stained with anti-CD3 antibody for 30 minutes at 4C and analyzed on an Attune Nxt flow cytometer (
Nucleofection of MG3-6/4 RNPs (104 pmol protein/120 pmol guide) comprising sgRNAs described below in Table 7D below and SEQ ID NOs: 383-572 was performed into Hep3B cells (100,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (
Nucleofection of MG3-6/4 RNPs (104 pmol protein/120 pmol guide) comprising sgRNAs described below in Table 7E below and SEQ ID NOs: 573-602 was performed into Hep3B cells (100,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (
To evaluate the ability of the MG3-6/3-4 chimeric Type II nuclease to edit the genome in vivo in a living animal, a lipid nanoparticle was used to deliver an mRNA encoding the MG3-6/3-4 nuclease (e.g. RNA version of SEQ ID NO: 603) and single guide RNAs (sgRNA) that target different parts of the coding sequence of the mouse HAO-1 gene (e.g. described in the tables below). The HAO-1 gene encodes glycolate oxidase which is an enzyme involved in glycolate metabolism and is expressed primarily in hepatocytes in the liver. A screen of sgRNAs that target the HAO-1 coding sequence was performed in the mouse liver cell line Hepa1-6 to identify active guides. The sgRNAs mH364-7 and mH364-20, which exhibited 46% and 26% editing in Hepa1-6 cells when transfected with the mRNA encoding the MG3-6/3-4 nuclease, were selected for testing in mice. mH364-7 targets exon 2 and mH364-20 targets exon 4.
A number of chemical modifications of the native RNA structure were incorporated into these sgRNAs. These chemical modifications were selected based on their ability to improve the stability of the sgRNA in vitro when incubated in extracts from mammalian cells without negatively impacting editing activity. For initial testing in mice, sgRNAs mH364-7 and mH364-20 incorporating chemistry 1 and chemistry 35 were selected for testing and designated as mH364-7-1, mH364-20-1, mH364-7-35, mH364-20-35. The sequences of these guides including the chemical modifications are shown below in Table 9.
The mRNA encoding the MG3-6/3-4 nuclease was generated by in vitro transcription of a linearized plasmid template using T7 RNA polymerase, nucleotides, and enzymes purchased from New England Biolabs or Trilink Biotechnologies.
The DNA sequence (SEQ ID No: 603) that was transcribed into RNA comprised the following elements in order from 5′ to 3′: the T7 RNA polymerase promoter, a 5′ untranslated region (5′ UTR), a nuclear localization signal, a short linker, the coding sequence for the MG3-6/3-4 nuclease, a short linker, a nuclear localization signal, and a 3′ untranslated region and an approximately 100 nucleotide polyA tail (not included in SEQ ID No: 603).
The protein sequence encoded in the synthetic mRNA encoded in this MG3-6/3-4 cassette comprises the following elements from 5′ to 3′: the nuclear localization signal from SV40, a five amino acid linker (GGGS), the protein coding sequence of the MG3-6/3-4 nuclease from which the initiating methionine codon was removed, a 3 amino acid linker (SGG) and the nuclear localization signal from nucleoplasmin. The DNA sequence of the protein coding region of this cassette was modified to reflect the codon usage in humans using a commercially available algorithm. An approximately 100-nucleotide polyA tail was encoded in the plasmid used for in vitro transcription and the mRNA was co-transcriptionally capped using the CleanCAP™ reagent purchased from Trilink Biotechnologies. Uridine in the mRNA was replaced with N1-methyl pseudouridine.
The lipid nanoparticle (LNP) formulation used to deliver the MG3-6/3-4 mRNA and the guide RNA is based on LNP formulations described in the literature including Kauffman et al (Nano Lett. 2015, 15, 11, 7300-7306 (https://doi.org/10.1021/acs.nanolett.5b024970). The four lipid components were dissolved in ethanol and mixed in an appropriate molar ratio to make the lipid working mix. The mRNA and the guide RNA were either mixed prior to formulation at a 1:1 mass ratio or formulated in separate LNP that were later co-injected into mice at a 1:1 mass ratio of the two RNA's. In either case, the RNA was diluted in 100 mM Sodium Acetate (pH 4.0) to make the RNA working stock. The lipid working stock and the RNA working stock were mixed in a microfluidics device (Ignite NanoAssembler, Precision Nanosystems) at a flow rate ratio of 1:3, respectively and a flow rate of 12 mLs/min. The LNP were dialyzed against phosphate buffered saline (PBS) for 2 to 16 hours and then concentrated using Amicon spin concentrators (Millipore) until the reduced volume was achieved. The concentration of RNA in the LNP formulation was measured using the Ribogreen reagent (Thermo Fisher). The diameter and polydispersity (PDI) of the LNP were determined by dynamic light scattering. Representative LNP had diameters ranged from 65 nm to 120 nm with PDI of 0.05 to 0.20. LNP were injected intravenously into 8- to 12-week-old C57B16 wild type mice via the tail vein (0.1 mL per mouse) at a total RNA dose of 1 mg RNA per kg body weight. Eleven days post-dosing, 3 of the 5 mice in each group were sacrificed and the liver was collected and homogenized using a bead beater (Omni International) in a digestion buffer supplied in the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific). Genomic DNA was purified from the resulting homogenate using the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific) and quantified by measuring the absorbance at 260 nm. Genomic DNA purified from mice injected with buffer alone was used as a control. At 28 days post-dosing, the remaining 2 mice in each group were sacrificed and the liver was collected and homogenized using a bead beater (Omni International) in a digestion buffer supplied in the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific). Genomic DNA was purified from the resulting homogenate using the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific) and quantified by measuring the absorbance at 260 nm. Genomic DNA purified from mice injected with buffer alone was used as a control.
The liver genomic DNA was then PCR amplified using a first set of primers flanking the region targeted by the two guides. The PCR primers used are shown below in Table 10.
The 5′ end of these primers comprise conserved regions complementary to the PCR primers used in the second PCR, followed by 5 Ns in order to give sequence diversity and improve MiSeq sequencing quality, and end with sequences complementary to the target region in the mouse genome. PCR was performed using Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs) on 100 ng of genomic DNA and an annealing temperature of 60° C. for a total of 30 cycles. This was followed by a 2nd round of 10 cycles of PCR using primers designed to add unique dual Illumina barcodes (IDT) for next generation sequencing on a MiSeq instrument. Each sample was sequenced to a depth of greater than 10,000 reads using 150 bp paired end reads. Reads were merged to generate a single 250 bp sequence from which Indel percentage and INDEL profile was calculated using a proprietary Python Script.
The results of the NGS analysis of INDELS from mice at day 11 post dosing are shown in Table 11 for individual mice and are summarized in
Group 2 mice received LNP encapsulating guide RNA mH364-7-1. Group 3 mice received LNP encapsulating guide RNAmH364-7-35. Group 4 mice received LNP encapsulating guide RNA mH364-20-1. Group 5 mice received LNP encapsulating guide RNAmH364-20-35. All mice in groups 2 to 5 also received LNP encapsulating the MG3-6/3-4 mRNA that was mixed with the guide RNA containing LNP at a 1:1 RNA mass ratio prior to injection. No INDELS were detected in the liver of mice injected with PBS buffer (see Table 11). Mice injected with LNPs encapsulating guide 364mHA-G7-1 and MG3-6/3-4 mRNA exhibited INDELS at the target site in HAO-1 at a mean frequency of 53.0%. Mice injected with LNPs encapsulating guide 364mHA-G7-35 and MG3-6/3-4 mRNA exhibited INDELS at the target site in HAO-1 at a mean frequency of 23.6%. Mice injected with LNPs encapsulating guide 364mHA-G20-1 and MG3-6/3-4 mRNA exhibited INDELS at the target site in HAO-1 at a mean frequency of 8.9%. Mice injected with LNPs encapsulating guide 364mHA-G20-35 and MG3-6/3-4 mRNA exhibited indels at the target site in HAO-1 at a mean frequency of 2.5%. These data demonstrate that the guides with spacer 7 (364mHA-G7-1 and 364mHA-G7-35) are significantly more potent in vivo than the guides with spacer 20 (364mHA-G20-1 and 364mHA-G20-35) when guides with the same chemical modifications are compared. This is consistent with the higher level of editing observed with these 2 guide sequences in Hepa1-6 cells by mRNA-based transfection (mH364-7 exhibited 46% INDELS and mH364-20 26% INDELS in Hepa1-6 cells). Guide chemistry #1 resulted in higher levels of editing than chemistry #35 for both guide 7 (2.2-fold higher editing with chemistry #1) and guide 20 (3.5-fold higher editing with chemistry #1). These data demonstrate that the MG3-6/3-4 nuclease can edit in vivo in mice at the target site specified by the sgRNA. Moreover, an sgRNA with a set of chemical modifications designated chemistry #1 was able to promote editing at 53% of the genomic DNA in whole liver when delivered using an LNP. The LNP used in these studies is taken up via binding of apolipoprotein E (apoE) to the LNP which is a ligand for binding to the low-density lipoprotein receptor (see e.g. Yan et al, Biochem Biophys Res Commun 2005 328(1):57-62.doi: 10.1016/j.bbrc.2004.12.137, Akinc et al Mol Ther 2010 (7):1357-64, doi: 10.1038/mt.2010.85).
The liver is composed of a number of different cell types. In the liver of mice, the hepatocytes make up about 52% of all cells (and 35% of hepatocytes contain two nuclei), with Kupffer cells (18%), Ito cells (8%), and endothelial cells (22%) making up the remaining cells (Histochem Cell Biol 131, 713-726 https://doi.org/10.1007/s00418-009-0577-1). By extrapolation, without wishing to be bound by theory, about 60% [((52+(0.35×52))/(48+(52+(0.35×52)))] of the total nuclei in the mouse liver are predicted to be derived from hepatocytes. Because the LDL receptor is expressed mainly on hepatocytes in the liver (see e.g. https://www.proteinatlas.org/ENSG00000130164-LDLR/tissue/liver#imid_2815831), the LNP used in the mouse studies described herein is expected to be taken up primarily by hepatocytes. Because hepatocyte nuclei make up about 60% of all nuclei in the whole liver of mice, it can be predicted that if all the hepatocyte nuclei were edited, the level of INDELS measured in the whole liver are predicted to be about 60%. The finding that LNP delivery of MG3-6/3-4 was able to achieve INDEL rates of 53% suggests that the majority of hepatocyte nuclei were edited.
The HAO1 gene encodes the protein glycolate oxidase (GO), an intracellular enzyme involved in glycolate metabolism. To determine if the observed gene editing in the HAO1 gene resulted in a reduction in the expression of the GO protein in the liver, we extracted total protein from a separate lobe of the liver from mice in the same study. The GO protein was detected using a Western blot assay with commercially available antibodies against the mouse GO protein. The protein vinculin was used as a loading control on the Western blot, as Vinculin levels are predicted to not be impacted by gene editing of the HAO1 gene. As shown in
The impact of chemical modifications to the sgRNA upon in vivo editing efficiency was further investigated by testing 4 different guide chemistries introduced into the same guide RNA sequence. Guide RNA 7 that targets the mouse HAO1 gene was synthesized with chemical modifications #1, #35, #42, or #45. The sequences of these guides are shown below in Table 12.
The mRNA encoding MG3-6/3-4 nuclease was generated by in vitro transcription of a linearized plasmid template using T7 RNA polymerase, nucleotides, and enzymes purchased from New England Biolabs or Trilink Biotechnologies. The DNA sequence that was transcribed into RNA comprised the following elements in order from 5′ to 3′: the T7 RNA polymerase promoter, a untranslated region (5′ UTR), a nuclear localization signal, a short linker, the coding sequence for the MG3-6/3-4 nuclease, a short linker, a nuclear localization signal, and a 3′ untranslated region (SEQ ID No: 603) and an approximately 100 nucleotide polyA tail (not included in SEQ ID No: 603)
The protein sequence encoded in the synthetic mRNA encoded in this MG3-6/3-4 cassette comprises the following elements from 5′ to 3′: the nuclear localization signal from SV40, a five amino acid linker (GGGS), the protein coding sequence of the MG3-6/3-4 nuclease from which the initiating methionine codon was removed, a 3 amino acid linker (SGG), and the nuclear localization signal from nucleoplasmin. The DNA sequence of the protein coding region of this cassette was modified to reflect the codon usage in humans using a commercially available algorithm. An approximately 100 nucleotide polyA tail was encoded in the plasmid used for in vitro transcription, and the mRNA was co-transcriptionally capped using the CleanCAP™ reagent purchased from Trilink Biotechnologies. Uridine in the mRNA was replaced with N1-methyl pseudouridine. The lipid nanoparticle (LNP) formulation used to deliver the MG3-6/3-4 mRNA and the guide RNA is based on LNP formulations described in the literature including Kauffman et al (Nano Lett. 2015, 15, 11, 7300-7306, https://doi.org/10.1021/acs.nanolett.5b024970). The four lipid components were dissolved in ethanol and mixed in an appropriate molar ratio to make the lipid working mix. The mRNA and the guide RNA were either mixed prior to formulation at a 1:1 mass ratio or formulated in separate LNP that were later co-injected into mice at a 1:1 mass ratio of the two RNA's. In either case, the RNA was diluted in 100 mM Sodium Acetate (pH 4.0) to make the RNA working stock. The lipid working stock and the RNA working stock were mixed in a microfluidics device (Ignite NanoAssembler, Precision Nanosystems) at a flow rate ratio of 1:3, respectively, and a flow rate of 12 mLs/min. The LNP were dialyzed against phosphate buffered saline (PBS) for 2 to 16 hours and then concentrated using Amicon spin concentrators (Milipore) until the reduced volume was achieved. The concentration of RNA in the LNP formulation was measured using the Ribogreen reagent (Thermo Fisher). The diameter and polydispersity (PDI) of the LNP were determined by dynamic light scattering. Representative LNP had diameters ranged from 65 nm to 120 nm with PDI of 0.05 to 0.20. LNP were injected intravenously into 8- to 12-week-old C57B16 wild type mice via the tail vein (0.1 mL per mouse) at a total RNA dose of 1 mg RNA per kg body weight. Ten days post-dosing, 3 of the 5 mice in each group were sacrificed and the liver was collected and homogenized using a bead beater (Omni International) in a digestion buffer supplied in the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific). Genomic DNA was purified from the resulting homogenate using the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific) and quantified by measuring the absorbance at 260 nm. Genomic DNA purified from mice injected with buffer alone was used as a control. At 28 days post-dosing, the remaining 2 mice in each group were sacrificed and the liver was collected and homogenized using a bead beater (Omni International) in a digestion buffer supplied in the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific). Genomic DNA was purified from the resulting homogenate using the PureLink Genomic DNA Isolation Kit (Thermo Fisher Scientific) and quantified by measuring the absorbance at 260 nm. Genomic DNA purified from mice injected with buffer alone was used as a control.
The liver genomic DNA was then PCR amplified using a first set of primers flanking the region targeted by the two guides. The PCR primers used are shown in Table 10. The 5′ end of these primers comprise conserved regions complementary to the PCR primers used in the second PCR, followed by 5 Ns in order to give sequence diversity and improve MiSeq sequencing quality, and end with sequences complementary to the target region in the mouse genome. PCR was performed using Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs) on 100 ng of genomic DNA and an annealing temperature of 60° C. for a total of 30 cycles. This was followed by a 2nd round of 10 cycles of PCR using primers designed to add unique dual Illumina barcodes (IDT) for next generation sequencing on a MiSeq instrument. Each sample was sequenced to a depth of greater than 10,000 reads using 150 bp paired end reads. Reads were merged to generate a single 250 bp sequence from which Indel percentage and INDEL profile was calculated using a proprietary Python Script.
The editing results are summarized in
Control mice injected with PBS buffer did not contain measurable INDELS at the target site for guide 7. The mean INDEL frequency in mice that received LNP containing guides mH364-7-1, mH364-7-35, mH364-7-42, and mH364-7-45 was 46.1%, 26.6%, 32.4%, and 32.1%, respectively, demonstrating that guide RNA chemistry #1 was the most potent followed by #42 and #45, with chemistry #35 being the least potent. These data suggest that chemical modifications to the bases and backbone at the 5′ and 3′ ends of the guide RNA provided the highest in vivo potency amongst the chemistries tested. Additional modifications of internal bases did not improve in vivo potency. These findings are in contrast with published data for the spCas9 sgRNA where modifications of bases or the backbone at both the ends of the sgRNA and at internal sequences was required for optimal in vivo editing (Yin et al, Nature Biotechnology, doi:10.1038/nbt.4005) and modifications of just the 5′ and 3′ ends of the sgRNA enabled low levels of editing (20% INDELS) in the liver using delivery in a similar LNP.
Total RNA was purified from a separate lobe of the liver from the same mice described in Table 13 and used to measure level of HAO-1 mRNA by digital droplet PCR (dd-PCR). The PBS injected mice were used as controls and the levels of HAO-1 mRNA in the livers of edited mice were compared to these controls. The dd-PCR assay was designed and optimized using standard techniques. ddPCR is a highly accurate method for determining the absolute copy number of a specific nucleic acid in a complex mixture (e.g. Taylor et al Sci Rep 7, 2409 (2017). doi:10.1038/s41598-017-02217-x). The total liver RNA was first converted to cDNA by reverse transcription then quantified in the dd-PCR assay using GAPDH as an internal control to normalize between samples. As shown in Table 14, the level of HAO1 mRNA in the individual mice treated with LNP encapsulating MG3-6/3-4 mRNA and sgRNA targeting the mouse HAO1 gene was decreased, and the magnitude of decrease was correlated with the INDEL frequency.
The largest reduction in HAO1 mRNA was seen in the group of mice treated with sgRNA mH364-7-1, while the smallest reduction of HAO-1 mRNA was observed in mice treated with sgRNA mH364-7-35. A reduction in HAO1 mRNA can occur when frameshift mutations are introduced into the coding sequence of a gene via a mechanism called nonsense mediated decay (Brogna et al, Nat Struct Mol Biol 16, 107-113 (2009), doi:10.1038/nsmb.1550). The observation of reduced HAO-1 mRNA in the liver of mice edited at the HAO-1 gene with MG3-6/3-4 is consistent with the presence of INDELS that result in a high rate of frame shifts as shown in Table 15.
In Table 15, the mean frequency of INDELS that result in a frame shift in the HAO1 coding sequence were determined from the NGS data. This analysis shows that the majority of the INDELS resulted in a frameshift for all four of the sgRNA tested.
The HAO1 gene encodes the protein glycolate oxidase (GO) that is an intracellular enzyme involved in glycolate metabolism. To determine if the observed gene editing in the HAO1 gene resulted in a reduction in the expression of the GO protein in the liver, we extracted total protein from a separate lobe of the liver from mice in the same study described in
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application is a continuation of International Application No. PCT/PCT/US2022/013396 entitled “NOVEL ENGINEERED AND CHIMERIC NUCLEASES”, filed Jan. 21, 2022, which claims the benefit of U.S. Provisional Application No. 63/237,484, entitled “NOVEL ENGINEERED AND CHIMERIC NUCLEASES”, filed on Aug. 26, 2021, and U.S. Provisional Application No. 63/140,620 entitled “NOVEL ENGINEERED AND CHIMERIC NUCLEASES” filed on Jan. 22, 2021, each of which is incorporated by reference herein in its entirety. This application is related to International Application No. PCT/US2021/031136 entitled “ENZYMES WITH RUVC DOMAINS”, filed on May 6, 2021, and PCT/US2020/018432, filed on Feb. 14, 2020, entitled “ENZYMES WITH RUVC DOMAINS”, each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63140620 | Jan 2021 | US | |
| 63237484 | Aug 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/13396 | Jan 2022 | US |
| Child | 18056629 | US |
