US 2024/0355419 A1 · RAPID ASSEMBLY OF MULTIPLEX GRNA ARRAYS

Abstract

The present disclosure is directed to polycistronic guide RNAs, DNA encoding polycistronic gRNA, multiplex CRISPR vectors, a plurality of component DNA fragments for assembly into a DNA encoding a polycistronic gRNA array, a plurality of primer pairs for making a plurality of component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA, and methods of making multiplex CRISPR vectors. The current disclosure is directed to multiplexed CRISPR technologies that have great potential for pathway engineering and genome editing. In the current disclosure describes efficient assembly of tRNA/Csy4/Ribozyme-based gRNA arrays which can be produced in a quick and effective process.

Claims27

Dependent claims

A polycistronic guide RNA (gRNA) array, comprising:

nucleotide sequences of a plurality of guide RNAs (gRNAs) for use in a CRISPR-Cas system;
wherein:
a) each nucleotide sequence of a gRNA in the array:
i) comprises a gRNA targeting sequence and a gRNA binding sequence, wherein the gRNA targeting sequence in each nucleotide sequence of a gRNA is unique to that gRNA;
and the gRNA binding sequence is common to all the gRNAs in the array;
and
ii) is linked at the 5′ end to a common RNA cleavage recognition sequence (5′ RCRS);
and
b) upon cleavage by an RNA cleaving agent specific for the RNA cleavage recognition sequence, the polycistronic gRNA array generates the plurality of gRNAs.

6 more elements

A plurality of component DNA fragments for assembly into a DNA encoding a polycistronic gRNA array, wherein:

the total number of component DNA fragments is n+1, the total number of gRNAs in the polycistronic gRNA array is n, and n is equal or greater than 2;
the component DNA fragments are designated as the first to the (n+1)th component DNA fragments, and the gRNAs in the polycistronic gRNA array are designated as the first to the nth gRNA;
the DNA encoding a polycistronic gRNA array is generated when the component DNA fragments are assembled in the order of the first to the (n+1)th component DNA fragment in the 5′ to 3′ orientation, wherein:
a) the first component DNA fragment comprises, from 5′ to 3′:
i) the recognition sequence of a type IIS restriction enzyme,
ii) an upstream vector matching overhang sequence of variable length (e.g., 2, 3, 4, 5, or 6-bp),
iii) a nucleotide sequence encoding a common 5′ RNA cleavage recognition sequence (5′ RCRS),
iv) a nucleotide sequence encoding a 5′ portion of the targeting sequence of a first gRNA and comprising a downstream overhang sequence unique to the first gRNA;
and
v) the reverse complement of the recognition sequence of the type IIS restriction enzyme;
b) for each of the second to the (n+1)th component DNA fragments, with “p” representing a number from 2 to n, a pth component DNA fragment comprises, from 5′ to 3′:
i) the recognition sequence of the type IIS restriction enzyme,
ii) a nucleotide sequence comprising an upstream overhang sequence and encoding a 3′ portion of the targeting sequence of the (p−1)th gRNA, wherein the upstream overhang sequence is unique to the (p−1)th gRNA and complementary to the downstream (e.g., 2, 3, 4, 5, or 6-bp) overhang sequence in the (p−1)th component DNA fragment;
iii) a nucleotide sequence encoding a common gRNA binding sequence;
iv) a nucleotide sequence encoding the common 5′ RCRS,
v) a nucleotide sequence encoding a 5′ portion of the targeting sequence of the pth gRNA and comprising a downstream overhang sequence unique to the pth gRNA;
and
vi) the reverse complement of the recognition sequence of the type IIS restriction enzyme;
and
c) the (n+1)th component DNA fragment comprises, from 5′ to 3′:
i) the recognition sequence of the type IIS restriction enzyme;
ii) a nucleotide sequence comprising an upstream overhang sequence and encoding a 3′ portion of the targeting sequence of the nth gRNA, wherein the upstream overhang sequence is unique to the nth gRNA and complementary to the downstream overhang sequence in the nth component DNA fragment;
iii) a nucleotide sequence encoding the common gRNA binding sequence;
iv) a downstream vector matching nucleotide overhang sequence;
and
v) the reverse complement of the recognition sequence of the type IIS restriction enzyme.

23 more elements

A plurality of primer pairs for making a plurality of component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA array, wherein:

the total number of primer pairs is n+1, for making n+1 component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA array for n gRNAs, with n being equal or greater than 2;
the primer pairs are designated as the first to the (n+1)th primer pair, the component DNA fragments are designated as the first to the (n+1)th component DNA fragments, and the gRNAs in the polycistronic gRNA array are designated as the first to the nth gRNA;
the DNA encoding a polycistronic gRNA array is generated when the component DNA fragments are assembled in the order of the first to the (n+1)th component DNA fragment in the 5′ to 3′ orientation, wherein:
a) the first primer pair comprises a forward primer and a reverse primer, wherein:
the forward primer of the first primer pair comprises, from 5′ to 3′:
(i) the recognition sequence of a type IIS restriction enzyme;
(ii) an upstream vector matching overhang sequence;
and
(iii) a template specific sequence (e.g., a sequence encoding a 5′ portion of an RNA cleavage recognition sequence (RCRS);
and
the reverse primer of the first primer pair comprises, from 5′ to 3′:
(i) the recognition sequence of the type IIS restriction enzyme;
(ii) a sequence encoding a 5′ portion of the targeting sequence of the first gRNA and comprising a downstream overhang sequence unique to the first gRNA;
and
(iii) a template specific sequence (e.g., a sequence encoding a 3′ portion of the RNA cleavage recognition sequence (RCRS);
b) for each of the second to the (n+1)th primer pairs, with “p” representing a number from 2 to n, a pth primer pair comprises a forward primer and a reverse primer, wherein:
the forward primer of the pth primer pair comprises, from 5′ to 3′:
(i) the recognition sequence of the type IIS restriction enzyme,
(ii) a nucleotide sequence comprising an upstream overhang sequence and encoding a 3′ portion of the targeting sequence of the (p−1)th gRNA, wherein the upstream overhang sequence is unique to the (p−1)th gRNA and complementary to the downstream overhang sequence in the reverse primer of the (p−1)th primer pair, and
(iii) a template specific sequence (e.g., a sequence encoding a 5′ portion of a common gRNA binding sequence);
and
the reverse primer of the pth primer pair comprises, from 5′ to 3′:
(i) the recognition sequence of the type IIS restriction enzyme;
(ii) a sequence encoding a 5′ portion of the targeting sequence of the pth gRNA and comprising a downstream overhang sequence unique to the pth gRNA;
and
(iii) a template specific sequence (e.g., a sequence encoding a 3′ portion of an RCRS);
c) the (n+1)th primer pair comprises a forward primer and a reverse primer, wherein:
the forward primer the (n+1)th primer pair comprises, from 5′ to 3′:
(i) the recognition sequence of the type IIS restriction enzyme;
(ii) a nucleotide sequence comprising an upstream overhang sequence and encoding a 3′ portion of the targeting sequence of the nth gRNA, wherein the upstream overhang sequence is unique to the nth gRNA and complementary to the downstream (4-bp) overhang sequence in the reverse primer of the nth primer pair;
and
(iii) a template specific sequence (e.g., a sequence encoding a 5′ portion of the common gRNA binding sequence);
and
the reverse primer of the (n+1)th primer pair comprises, from 5′ to 3′;
(i) the recognition sequence of the type IIS restriction enzyme;
(ii) a downstream vector matching overhang sequence;
and
(iii) a template specific sequence (e.g., a sequence encoding a 3′ portion of the common gRNA binding sequence).

35 more elements

Dependencies are inferred from the claim text.

Rapid assembly of multiplex grna arrays

Life of this patent

Rapid assembly of multiplex grna arrays

Life of this patent

Abstract

Claims27