CRISPR Interference for Targeted Gene Silencing in Mycobacteria PDF

**CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria** **Abstract** The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close thi...

**CRISPR Interference (CRISPRi) for Targeted Gene Silencing in Mycobacteria** **Abstract** The genetic basis for Mycobacterium tuberculosis pathogenesis is incompletely understood. One reason for this knowledge gap is the relative difficulty of genetic manipulation of M. tuberculosis. To close this gap, we recently developed a robust CRISPR interference (CRISPRi) platform for programmable gene silencing in mycobacteria. In this chapter, we: (1) discuss some of the advantages and disadvantages of CRISPRi relative to more traditional genetic approaches; and (2) provide a protocol for the application of CRISPRi to reduce transcription of target genes in mycobacteria. **Introduction** The past 20years have seen dramatic improvements in our ability to genetically manipulate Mycobacterium tuberculosis. These improvements have advanced our understanding of this pathogen. While powerful, each genetic method has advantages and disadvantages. For example, current genetic approaches in M. tuberculosis include promoter replacement and inducible protein degradation systems that allow the regulation of target protein levels over two orders of magnitude \[1--3\], but can take months to target a single gene. Similarly, several methodologies \[4, 5\] now allow for facile gene deletion in M. tuberculosis, but these methods are necessarily restricted to the analysis of genes nonessential for in vitro growth and remain slow to implement. To increase throughput, Kenan Murphy and colleagues developed ORBIT \[6\], a method that dramatically improves the efficiency of M. tuberculosis genetic engineering. While it is a major advance, this method in its present form does not yet scale to parallelized genome-wide genetic manipulation. To work at scale, Transposon-sequencing (TnSeq) allows the simultaneous assessment of hundreds of thousands of loss-of function mutants. But as with gene deletion, TnSeq as currently implemented in M. tuberculosis is limited to analysis of genes non essential for in vitro growth \[7, 8\]. Lastly, none of these methods provide a simple mechanism to simultaneously modulate multiple genes in order to elucidate genetic interactions. To complement existing genetic tools, we recently developed an optimized CRISPR interference (CRISPRi) system for targeted gene silencing in mycobacteria \[9\] (Fig. 1). Unlike most other CRISPRi applications which utilize a Cas9 enzyme derived from Streptococcus pyogenes (SpyCas9) \[10, 11\], we found a Cas9 enzyme derived from S. thermophilus (Sth1Cas9) to have superior performance characteristics (magnitude of target gene knockdown and reduced toxicity) in Mycobacterium smegmatis \[9\]. In this system, the protein dCas9 (with two mutations that disable nuclease activity, thus "dead" or dCas9) is guided to the target gene by a chimeric RNA called a single guide RNA(sgRNA)\[12\]. Targeting specificity is determined both by base pairing of the sgRNA and target DNA, as well as a short DNA motif (protospacer adjacent motif \[PAM\]) within the target DNA sequence. The PAM is a 2--8 base pair sequence located immediately downstream of the sgRNA target sequence \[13--15\]. PAM recognition is an obligate first step for dCas9 binding---recognition of the PAM by dCas9 destabilizes the adjacent DNA duplex, thereby allowing interrogation of the DNA target by the sgRNA \[16, 17\]. Binding of the dCas9--sgRNA complex to the target gene results in transcriptional interference by blocking RNA polymerase promoter access or transcription elongation \[10, 11\]. The M. tuberculosis CRISPRi system \[9\] was further engineered to be inducible by two alternative small molecules (anhydrotetracycline or doxycycline), thereby allowing the facile manipulation of M. tuberculosis genes, be they essential or nonessential for in vitro growth. The efficient cellular and tissue penetration of doxycycline \[18\] should allow CRISPRi-mediated control of the M. tuberculosis transcriptome in numerous experimental settings, including axenic in vitro culture, ex vivo M. tuberculosis infected macrophages, and in vivo animal infection models. An additional advantage of the Sth1 dCas9 CRISPRi system is that the magnitude of target gene silencing is tunable, either by varying targeted PAM "strength" \[9\] or by varying the length of the sgRNA targeting sequence \[10\]. This allows for rheostat-like control of target gene production spanning two orders of magnitude \[9, 10\]. Tunability enables the hypomorphic or partial silencing of target gene production to create an allelic series, thereby enabling the study of interactions (chemical and genetic) between in vitro essential genes \[9, 19\]. Lastly, CRISPRi is scalable. With advances in array-based synthesis, generating large pools of unique sgRNA targeting sequences is fast and inexpensive. Altogether, these unique features set the stage to develop CRISPRi as a powerful new genetic method in M. tuberculosis. In the next sections, we discuss the application and limitations of this approach for targeted gene silencing in M. tuberculosis.