FIELD

The present disclosure relates generally to Clustered regularly interspaced short palindromic repeat (CRISPR) systems, and more particularly to compositions and methods involving an improved Cas9 nuclease with enhanced spacer acquisition properties.

BACKGROUND

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins protect bacteria and archaea against their viruses (Barrangou et al., 2007) and plasmids (Marraffini and Sontheimer, 2008). In the first step of the CRISPR immune response, a very low proportion of the infected cells acquire a short sequence, known as a spacer sequence, of the invading genome in between the repeats of the CRISPR array (Barrangou et al., 2007). Spacer acquisition is catalyzed by the Cas1/Cas2 integration complex (Nunez et al., 2014; Nunez et al., 2015; Yosef et al., 2012) and results in the immunization of the host (Barrangou et al., 2007). In the second step of the CRISPR immune response, spacer sequences are transcribed and processed into a small RNA known as the CRISPR RNA (crRNA) (Brouns et al., 2008; Carte et al., 2008; Deltcheva et al., 2011). The crRNA is used as a guide by Cas nucleases to find its complementary sequence, known as the protospacer, in the invading viral or plasmid genome (Gasiunas et al., 2012; Jinek et al., 2012; Jore et al., 2011; Samai et al., 2015). Target recognition through base-pairing between the crRNA and the target DNA results in the destruction of the invader and host immunity (Garneau et al., 2010).

Based on their cas genetic repertoire, CRISPR-Cas systems have been classified into six types, I through VI (Makarova et al., 2015; Shmakov et al., 2015). Cas9 is the crRNA-guided nuclease of the type II-A CRISPR-Cas system ofStreptococcus pyogenes(Jinek et al., 2012). In addition to protospacer recognition by the crRNA, Cas9 target cleavage requires a 5′-NGG-3′ protospacer adjacent motif (PAM) immediately downstream of the target (Anders et al., 2014; Deveau et al., 2008; Jiang et al., 2013; Jinek et al., 2012). Cas9 is also required for the immunization step of the CRISPR response (Heler et al., 2015; Wei et al., 2015), using its PAM binding domain to specify functional spacer sequences that are flanked by the required NGG motif (Heler et al., 2015). In support of its role in spacer acquisition, Cas9 can associate in vivo with the other proteins encoded by the type II-A CRISPR-Cas system: Cas1, Cas2 and Csn2 (Heler et al., 2015). Cas9 systems have been utilized in a wide variety of compositions and methods, but there is an ongoing and unmet need for improvements in such systems, and methods of using them. This disclosure is pertinent to these needs.

SUMMARY

The present disclosure relates to a novel Cas9 enzyme comprising mutations that enhance its properties, relative to un-mutated Cas9. In particular, the altered Cas9 enzymes of this disclosure exhibit i) an increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG PAMs, or a combination of i) and ii). The altered Cas9 enzymes comprise an amino acid substitution of I473 and K500 in aStreptococcus pyogenesCas9 enzyme, one non-limiting example of which is provided in SEQ ID NO:1 as a non-mutated sequence, but other homologous changes can be made in other Cas9 enzymes. Thus, in embodiments, novel Cas9 enzymes of this disclosure comprise sequences that are at least 80% similar to SEQ ID NO:1 across its length, but retain one or more of the increased rate of spacer acquisition, or increased cleavage efficiency.

The disclosure includes polynucleotides, including but not limited to expression vectors, that encode the Cas9 enzymes described herein, cells comprising such polynucleotides, and methods of using such cells for a variety of purposes, such as for use in labelling bacteria, and for enhancing phage acquired immunity in bacteria, such as those used in industrial processes, including but not necessarily limited to the food a beverage industry, such as the dairy industry.

The disclosure includes methods of making modified bacteria by introducing into them expression vectors that encode the novel Cas9 enzymes described herein, and includes the modified cells, their cell culture medium, cell lysates, and Cas9 enzymes isolated from the cells.

In one approach the disclosure provides a method comprising contacting bacteria that have been modified to express a Cas9 described herein with one or more bacteriophage such that at least one spacer sequence in the genome of the bacteriophage is acquired by the bacteria. Spacer acquisition is more efficient than compared to a reference, such as an unmodified Cas9, i.e., a Cas9 that does not contain the described mutations. In certain embodiments, the bacteria are contacted with a plurality of distinct bacteriophage, and the bacteria acquire a plurality of distinct spacer sequences. In such implementations, the bacteriophage can be obtained from any source, including but not limited to a bacterial culture that is used in connection with making or finishing a food or beverage product. Such food products made with the assistance of modified bacteria are included within this disclosure.

