US 2021/0172008 A1 · METHODS AND COMPOSITIONS TO IDENTIFY NOVEL CRISPR SYSTEMS

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.

Claims30

Dependent claims

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.

3 more elements

Dependencies are inferred from the claim text.

Methods and compositions to identify novel crispr systems

Life of this patent

Methods and compositions to identify novel crispr systems

Life of this patent

Abstract

Claims30