# US 2021/0172008 A1 · Methods and compositions to identify novel crispr systems

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- Number: US 2021/0172008 A1
- Document ID: US20210172008A1
- Status: Published application
- Kind: A1
- Application number: 17/045,053
- Filed: 2019-04-03
- Published: 2021-06-10
- Earliest claimed: 2018-04-04
- Assignees: LifeEDIT, Inc.
- Applicants: Lifeedit, Inc.
- Inventors: Alexandra Briner Crawley; James R. Henriksen; Mark Moore; Rebecca E. Thayer
- Web page: https://patentgrep.com/patent/US20210172008A1

## CPC

- C07K 14/195: from bacteria
- C12Q 1/6813: Hybridisation assays
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions and methods for isolating new variants of known clustered regularly-interspaced short palindromic repeat (CRISPR) RNA-guided nuclease (RGN) genes and new CRISPR systems are provided. The methods find use in identifying CRISPR RGN gene variants in complex mixtures. Compositions comprise hybridization baits that hybridize to CRISPR RGN genes of interest in order to selectively enrich variant polynucleotides from complex mixtures. Bait sequences may be specific for a number of CRISPR RGN genes from distinct gene families of interest and may be designed to cover each CRISPR RGN gene of interest by at least 2-fold. Bait pools may also comprise baits for sequences flanking CRISPR RGN genes of interest to allow for the identification of tracrRNAs corresponding to novel CRISPR RGN variants and a complete CRISPR system comprising a CRISPR RGN and its associated guide RNA.

## Claims

### Claim 1

A method for identifying a variant of a clustered regularly-interspaced short palindromic repeat (CRISPR) RNA-guided nuclease (RGN) gene of interest comprising:
a) preparing DNA for hybridization from a complex sample comprising a variant of a CRISPR RGN gene of interest, thereby forming a prepared sample DNA comprising said variant of said CRISPR RGN gene of interest;
b) mixing said prepared sample DNA with a labeled bait pool comprising polynucleotide sequences complementary to said CRISPR RGN gene of interest;
c) hybridizing said prepared sample DNA to said labeled bait pool under conditions that allow for hybridization of a labeled bait in said labeled bait pool with said variant of said CRISPR RGN gene of interest to form one or more hybridization complexes comprising captured DNA;
d) sequencing said captured DNA; and
e) analyzing said sequenced captured DNA to identify said variant of said CRISPR RGN gene of interest.

### Claim 2 (depends on 1)

The method of claim 1, wherein said complex sample is an environmental sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein said complex sample is a mixed culture of at least two organisms.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The method of claim 1, wherein said labeled baits are specific for at least 10 CRISPR RGN genes of interest.

### Claim 6 (depends on 5)

The method of claim 5, wherein said labeled baits are specific for at least 300 CRISPR RGN genes of interest.

### Claim 7 (depends on 1)

The method of claim 1, wherein said labeled bait pool comprises at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, or at least 50,000 labeled baits.

### Claim 8 (depends on 1)

The method of claim 1, wherein at least 50 distinct labeled baits are mixed with said prepared sample DNA.

### Claim 9 (depends on 1)

The method of claim 1, wherein said labeled baits are 50-200 nt, 70-150 nt, 100-140 nt, or 110-130 nt in length.

### Claim 10 (depends on 1)

The method of claim 1, wherein said labeled baits comprise overlapping labeled baits, said overlapping labeled baits comprising at least two labeled baits that are complementary to a portion of a CRISPR RGN gene of interest, wherein the at least two labeled baits comprise different DNA sequences that are overlapping.

### Claim 11 (depends on 10)

The method of claim 10, wherein at least 10, at least 30, at least 60, at least 90, or at least 120 nucleotides of each overlapping labeled bait overlap with at least one other overlapping labeled bait.

### Claim 12 (depends on 1)

The method of claim 1, wherein said prepared sample DNA is enriched prior to mixing with said labeled baits.

### Claim 13 (depends on 1)

The method of claim 1, wherein said one or more hybridization complex is captured and purified from unbound prepared sample DNA.

### Claim 14 (depends on 13)

The method of claim 13, wherein said one or more hybridization complex is captured using a binding partner of said label of said labeled baits attached to a solid phase.

### Claim 15

(canceled)

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The method of claim 1, wherein captured DNA from said one or more hybridization complex is amplified and index tagged prior to said sequencing.

### Claim 18

(canceled)

### Claim 19

(canceled)

### Claim 20 (depends on 1)

The method of claim 1, wherein said analyzing said sequenced captured DNA comprises performing a sequence similarity search using the sequenced captured DNA against a database of known CRISPR RGN sequences or domains.

### Claim 21

(canceled)

### Claim 22

(canceled)

### Claim 23 (depends on 1)

The method of claim 1, wherein said labeled bait pool further comprises polynucleotide sequences complementary to sequences flanking said CRISPR RGN gene of interest, and wherein said method further comprises analyzing said sequenced captured DNA for sequences flanking said variant CRISPR RGN gene to identify a sequence encoding a tracrRNA of said variant of said CRISPR RGN gene of interest.

### Claim 24 (depends on 23)

The method of claim 23, wherein said flanking sequences comprise about 180 nucleotides on either side of said CRISPR RGN gene of interest.

### Claim 25

(canceled)

### Claim 26 (depends on 23)

The method of claim 23, wherein analyzing said flanking sequences comprises performing a sequence similarity search using the flanking sequences against a database of known CRISPR tracrRNA sequences.

### Claim 27

(canceled)

### Claim 28 (depends on 1)

The method of claim 1, wherein said method further comprises assaying a guide RNA comprising a crRNA for binding between the guide RNA and said variant of said CRISPR RGN gene of interest.

### Claim 29 (depends on 28)

The method of claim 28, wherein said method further comprises identifying a protospacer adjacent motif (PAM) and assaying said variant of said CRISPR RGN gene of interest and said guide RNA for binding to a target nucleotide sequence of interest adjacent to said PAM.

### Claim at position 30

30-47. (canceled)