# US 11,898,141 B2 · High-throughput assembly of genetic elements

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- Number: US 11,898,141 B2
- Document ID: US11898141B2
- Status: Granted
- Kind: B2
- Application number: 15/313,863
- Filed: 2015-05-27
- Published: 2017-09-21
- Granted: 2024-02-13
- Earliest claimed: 2014-05-27
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Inventors: Robert Nicol; Lauren Andrews; Tarjei Mikkelsen; Christopher Voigt
- Web page: https://patentgrep.com/patent/US11898141B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/20: Sequence assembly
- B01J 2219/00722: Nucleotides
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

Disclosed is a method of generating a set of sequence-verified nucleic acid elements for the combinatorial construction of genetic elements. The method includes: providing a plurality of nucleic acid parts; assembling nucleic acid parts to form a one or more nucleic acid elements, wherein the nucleic acid elements include at least two sequences selected from the plurality of parts; and determining the sequence of the nucleic acid elements. Further disclosed is a pool of higher-order nucleic acid constructs or amplification products thereof, comprising one or more nucleic acid elements as well as kits including a pool of sequence-verified nucleic acid elements of claims and/or a pool of higher-order nucleic acid constructs; and a plurality of primers for retrieving one or more sequence-verified nucleic acid elements and/or higher-order nucleic acid constructs.

## Claims

### Claim 1

A set of sequence-verified nucleic acid elements for combinatorial construction of genetic elements, said set comprising two or more nucleic acid elements assembled from a plurality of nucleic acid parts, comprising one or more categories of nucleic acid parts, wherein the nucleic acid elements comprise:
at least two sequences selected from the plurality of nucleic acid parts;
(ii) a 3′ flanking nucleic acid sequence and a 5′ flanking nucleic acid sequence, which together are capable of functioning to order assembly of higher-order nucleic acid constructs, to facilitate retrieval, and to identify the nucleic acid element,
wherein the 3′ and 5′ flanking sequences comprise one or more nucleic acid barcodes that are unique to each sequence-verified nucleic acid element, which together are capable of functioning to facilitate retrieval and to identify the nucleic acid element, and
wherein the 3′ and 5′ flanking sequences comprise one or more restriction sites that are capable of being cleaved prior to assembly in order to produce sticky ends to facilitate ordered assembly of higher order nucleic acid elements; and
(iii) at least one DNA hash,
wherein each DNA hash comprises a unique nucleic acid barcode capable of functioning to identify either the nucleic acid element or a nucleic acid part in the nucleic acid element,
wherein the barcode of each DNA hash is flanked by primer binding sites capable of being amplified to produce an amplicon capable of forming a concatemer with an amplicon from another DNA hash in another nucleic acid element,
wherein the primer binding sites are different for nucleic acid elements in the set of sequence-verified nucleic acid elements that are for each position within a combinatorial construction of genetic elements; and
wherein each DNA hash is located internally to the cleavage sites for the one or more restriction sites in (ii), whereby the hash cannot be removed by restriction enzyme digestion at the one or more restriction sites.

### Claim 2 (depends on 1)

The set of nucleic acid elements of claim 1, wherein at least one of the barcodes further comprise one or more sequencing adaptors, universal priming sites, sequence specific priming sites to facilitate retrieval, indexes, sequences that enable capture and/or amplification, one or more sequences that enable library construction, or a combination thereof.

### Claim 3 (depends on 1)

The set of nucleic acid elements of claim 1, wherein the nucleic acid parts comprise one or more sequence variants, orthologs or homologs; or
wherein the plurality of nucleic acid parts comprises one or more of:
a set of nucleic acid sequences comprising different promoters;
a set of nucleic acid sequences comprising a set of different ribosomal binding sites;
a set of nucleic acid sequences encoding a polypeptide;
a set of nucleic acid sequences encoding one or more non-coding RNAs; and/or
a set of nucleic acid sequences comprising a set of different transcription terminators.

### Claim 4 (depends on 3)

The set of nucleic acid elements of claim 3, wherein the nucleic acid elements comprise at least three sequences selected from the set of promoters, ribosomal binding sites, sequences encoding a polypeptide, sequences encoding one or more non-coding RNAs, and transcription terminators; or
wherein the nucleic acid elements comprise at least four sequences selected from the set of promoters, ribosomal binding sites, sequences encoding a polypeptide, sequences encoding one or more non-coding RNAs, and transcription terminators.

### Claim 5 (depends on 1)

A set of plasmids comprising the set of sequence-verified nucleic acid elements of claim 1.

### Claim 6 (depends on 1, 5)

A kit comprising:
the set of sequence-verified nucleic acid elements of claim 1; or
(ii) the set of plasmids of claim 5.

### Claim 7 (depends on 6)

The kit of claim 6, further comprising a plurality of primers for retrieving one or more of the sequence-verified nucleic acid elements, wherein the primers are specific for the unique nucleic acid barcodes.

### Claim 8 (depends on 1)

The set of nucleic acid elements of claim 1, wherein the nucleic acid elements comprise a cistron.

### Claim 9 (depends on 1)

The set of nucleic acid elements of claim 1, wherein the one or more restriction sites comprise at least one Type IIs restriction site.

### Claim 10 (depends on 1)

A method of optimizing a combination of nucleic acid elements for production of an optimum desired phenotype, the method comprising:
retrieving a plurality of sequences of:
the set of sequence-verified nucleic acid elements of claim 1; and/or
(ii) a pool of higher-order nucleic acid constructs comprising the sequence-verified nucleic acid elements of claim 1;
assembling the plurality of retrieved sequences to construct a biological pathway assembly;
testing each of the assembled biological pathway assemblies fora desired phenotype;
determining which characteristic or characteristics of the assembled biological pathway contributes to the desired phenotype by comparing multiple assemblies; and
repeating the retrieving, assembling, testing, and determining until an assembled biological pathway is identified that produces said optimum desired phenotype.