Harnessing CRISPR-Cas9 for genome editing inStreptococcus pneumoniae

Abstract

13CRISPR systems provide bacteria and archaea with adaptive immunity against viruses and 14 plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system 15 can be exploited as a biotechnological tool for precise genome editing at a targeted locus. 16Here, we developed a novel, replicative plasmid that carries the CRISPR-Cas9 system for 17 RNA-programmable, genome editing by counterselection in the opportunistic human 18 pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making 19 targeted, marker-less gene knockouts and large genome deletions. After a precise double-20 stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed 21 repair (HDR) pathway is being exploited to select successful transformants. This is achieved 22 through the transformation of a template DNA fragment that will recombine in the genome and 23 eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered 24 strain, can be easily cured from the plasmid that is temperature-sensitive for replication, by 25 growing it at the non-permissive temperature. This allows for consecutive rounds of genome 26 editing. Using this system, we engineered a strain with three major virulence factors deleted. 27The here developed approaches should be readily transportable to other Gram-positive 28 bacteria. 29 30 31 65 crRNA that is base-paired to a trans-activating crRNA (tracrRNA) forms a two-RNA structure 66 that directs the CRISPR-associated proteins (e.g. Cas9) to introduce a double-stranded break 67 4 (DSB) into the target DNA locus. Site-specific cleavage occurs at locations determined by both 68 base-pairing complementarity between the crRNA and the target protospacer DNA and a short 69 protospacer adjacent motif (PAM) (Jinek et al. 2012). It has been demonstrated that the 70 endonuclease can be programmed by engineering the mature dual-tracrRNA: crRNA as a 71 single RNA chimera (sgRNA for single guide RNA), to cleave specific DNA sites. Thereby, 72 modified versions of the system can be exploited as a biotechnological tool for precise, RNA-73 programmable genome targeting and editing (Jinek et al. 2012). 74After the DSB has been introduced, the cell can utilize two major pathways in order to 75 repair the break and survive: homologous recombination (HR) or non-homologous end-joining 76 (NHEJ). In HR, a second intact copy of the broken chromosome segment, homologous to the 77 DSB site, serves as a template for DNA synthesis across the break. In this mechanism, the 78 crucial process of locating and recombining the homologous sequence is performed by RecA 79 (Shuman and Glickman 2007). NHEJ does not rely on a homologous DNA template, as the 80 two DNA ends are rejoined directly together. Most bacteria such as S. pneumoniae cannot 81 perform NHEJ, while it is capable to perform HR (Prudhomme et al. 2002; 2014). DSB repair 82 can be used as a way to generate mutants or desired changes to the genome by providing a 83 HR template, and forms ...

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq

“…These integrative plasmids were then transformed into S. pneumoniae . Transformation was performed with cells that were made competent by addition of the Competence Stimulating Peptide (CSP-1) using a previously published protocol 77 . Primers that were used for cloning are listed in Table 3.…”