Tagged microorganisms and methods of tagging

摘要

The present invention provides methods for tagging and/or identifying microorganisms. In some preferred embodiments, the microorganisms are bacteria. In some particularly preferred embodiments, the bacteria are members of the genus Streptococcus, while in other embodiments, the bacteria are members of other genera. The present invention also provides microorganisms tagged using the methods set forth herein. In some preferred embodiments, the tagged microorganisms are bacteria. In some particularly preferred embodiments, the tagged bacteria are members of the genus Streptococcus, while in other embodiments, the tagged bacteria are members of other genera.

主权项

1. A method for generating at least one CRISPR variant comprising a tag, comprising the steps of:
(a) exposing a parent bacterium comprising a CRISPR locus and a cas gene to at least one bacteriophage to produce a culture of bacteriophage resistant variant bacteria comprising a modified CRISPR locus, under conditions such that said modified CRISPR locus comprises an additional repeat-spacer unit, naturally inserted therein, wherein the spacer of said additional repeat-spacer unit has a length of between about 20 bp and about 58 bp and has 100% identity to a nucleotide sequence in the genome of said at least one bacteriophage wherein said additional repeat-spacer unit provides a tag; (b) selecting said bacteriophage resistant variant bacteria; (c) comparing said CRISPR locus or a portion thereof of said parent bacterium and said modified CRISPR locus of said bacteriophage insensitive variant bacteria, to identify bacteriophage insensitive variant bacteria comprising in their modified CRISPR locus, an additional repeat-spacer unit, is absent from said CRISPR locus of said parent bacterium; and (d) selecting said bacteriophage insensitive variant bacteria comprising in their modified CRISPR locus an additional repeat-spacer unit, wherein said additional repeat-spacer unit is not present in the parent bacterium; and (e) isolating and/or cloning and/or sequencing the additional repeat-spacer unit.