CRISPR technologies : a toolkit for making genetically intractable microbes tractable

Genetic manipulation of microbial species has been critical in dissecting their biology — yet many microbial species lacked robust tools for comprehensive genetic analysis until the advent of CRISPR-based techniques. Here, we review CRISPR-related advances that have enabled genome engineering and genetic analysis of difficult-to-manipulate microbial organisms, with an emphasis on mycobacteria, fungi, and parasites. We discuss how CRISPR-based analyses in these organisms have uncovered novel gene functions, dissected genetic interaction networks, and identified virulence factors. Introduction Microbial species represent the most abundant and diverse organisms on Earth, with critical roles in environmental homeostasis, industrial manufacturing, agriculture, and human health and disease. Unravelling the complex biology of these microorganisms has largely been dependent on our ability to manipulate them genetically. Genetic analysis of microbial organisms has a long history of pioneering and innovative experiments, including the formative discovery of transformation in ​Streptococcus pneumoniae​1​ and the subsequent identification of DNA as the critical carrier of genetic information​2​. The earliest accounts of genetic engineering involved the generation of transgenic ​Escherichia coli​ lineages through transformation of a recombinant plasmid encoding an antibiotic-resistance gene​3​. Since then, genetic manipulation of microbial organisms has been critical for the development of new biotechnologies and the study of microbial organisms themselves. Recent advances in molecular techniques have improved our ability to perform genetic manipulation in diverse microbial organisms. Modern technology platforms currently exist for functional genomic analysis and systems-level forward and reverse genetics in many microbial species, particularly model organisms. Such platforms include genome-wide genetic deletion libraries in ​E. coli​4​, whole-genome single-deletion​5​ and double-deletion​6​ libraries in the model yeast ​Saccharomyces cerevisiae​, and transposon sequencing for systems-level genetic analysis of several bacterial species​7​, including ​Salmonella​ ​enterica​8​ and ​Pseudomonas aeruginosa​9​. These biotechnologies have enabled large-scale genetic analyses to assess gene function and identify genetic interactions. Despite advances in technologies for systems-level functional genomic analysis in many microbial species, other microorganisms have remained relatively difficult to engineer genetically, which has hindered our potential to unlock their secrets. New advances in genomic manipulation — particularly CRISPR-Cas-based tools — have revolutionized our ability to perform targeted genetic manipulations in a diversity of organisms and been instrumental in enabling us to alter the genomes of even the most notoriously intractable microbial species. CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, are a group of DNA sequences in prokaryotes that play an important role in bacterial and archaeal immunity, and have been co-opted for genome editing across the tree of life​10,11​. In type II CRISPR systems, “spacer” sequences from CRISPR loci (derived from viral, plasmid or other foreign DNA which have been inserted into the CRISPR locus) are transcribed into RNA, and processed into small CRISPR RNAs (crRNAs), which then pair with CRISPR-associated (Cas) proteins and a trans-activating crRNA (tracrRNA). Together, this complex is targeted to “protospacer” sequences based on complementarity to the crRNA sequence and the presence of a protospacer adjacent motif (PAM) site. Cas proteins are endonucleases that cause double-strand breaks (DSBs) at the target protospacer locus. While this system is used by prokaryotes to detect and cleave invading foreign DNA, it can also be exploited as a biotechnology tool for precise genome editing at a targeted locus. This was first demonstrated using the Cas9 protein from ​Streptococcus pyogenes​ and a modified chimeric single-guide RNA (sgRNA), which fused together the crRNA and tracrRNA. By manipulating the sequence of this sgRNA, Cas9 could be programmed to target specific DNA sequences for cleavage, generating a DSB​10​. While sgRNA targeting is relatively flexible, the requirement of a PAM sequence can limit the availability of genomic target sites. Furthermore, organisms with extreme skew in GC or AT richness will influence whether or not a particular PAM is likely to be present at a high frequency. Once a DSB is generated, the manner in which it is repaired is responsible for the observed genome editing outcomes: breaks can be repaired by non-homologous end joining (NHEJ) that can result in insertions or deletions (indels) at the target locus, by alternative NHEJ pathways such as microhomology-mediated end joining that can result in genetic mutations, deletions, and translocations, or by homology-directed repair (HDR) if a donor DNA template with homology to the target locus is supplied. The latter strategy allows for precise mutations or alterations at the target locus. A property of Cas9 targeting in many microbial species is that the generated DSBs tends to be poorly repaired by NHEJ mechanisms, despite the presence of such machinery within the genome of the targeted organism​12​. In the case where a homologous donor is simultaneously provided, Cas9 is able to negatively select against any unmodified cells when targeted to a genomic locus​13​. In contrast, if Cas9 is directed towards a episomal plasmid the element will be lost from the resulting population of Cas9 expressing cells​14,15​. Newer variants of this technology such as CRISPR interference (CRISPRi) exploit a nuclease-dead version of the Cas9 enzyme (dCas9) targeted to specific genomic loci by sgRNAs to achieve steric hindrance of RNA polymerase, thus blocking transcription initiation or elongation​16–18​. Together, these groundbreaking technologies have been used to alter the sequence and modulate the expression of genes in a remarkably wide variety of species​11​. Here we review advances in CRISPR-based techniques for rigorous genetic analysis of otherwise difficult-to-work-with microbial organisms. We describe challenges and limitations associated with using traditional methods for genetic manipulation, and highlight how CRISPR-based technologies are helping to overcome these biological and technological hurdles. We focus on recent developments in CRISPR-based techniques for the analysis of genetically unwieldy microbial organisms, including mycobacteria, microbial fungi, and eukaryotic parasites, and explain how CRISPR-based work in these organisms has been instrumental for generating genetic mutants and performing functional genomic analysis, dissecting genetic interaction networks, and conducting complex genome engineering. We close by discussing how ongoing technological advances in CRISPR-based platforms will undoubtedly yield exciting new capabilities for the study of the microbial world. Generating microbial mutants with CRISPR A critical component of functional genomic analysis is the ability to generate genetic mutations or deletions, or otherwise knock-down gene function so as to assess resultant phenotypes. This reverse genetic analysis strategy has been instrumental in dissecting genetic perturbations and understanding gene function in many microbes. However, similar analysis in genetically unwieldy microbial organisms has lagged behind, due to limitations associated with generating genetic perturbations in such organisms. A common biological limitation amongst intractable microorganisms is inefficient homologous recombination, which is often needed for classic genetic manipulation techniques. Low rates of homologous recombination and the requirement for very long stretches of homologous sequence for effective recombination have hindered the generation of genetic mutants in many mycobacterial​19,20​, fungal​21–23​, and parasitic microbes​24–27​. For targeted gene disruption, CRISPR-based editing can bypass the need to use traditional homologous recombination-based approaches, since CRISPR-Cas-induced DSBs can be repaired via NHEJ, in a manner that is mutagenic to the target locus and independent of homologous recombination (Figure 1a). To introduce a precise genetic alteration, CRISPR-Cas-based editing systems can improve the efficiency of homologous recombination-based genome editing through the generation of a genomic DSB and stimulating homologous recombination at the locus of interest​24,28–31​. Additionally, CRISPRi can bypass the need to make targeted mutations in the genome, by enabling users to simply “knock down” gene expression for their target of interest (Figure 1b). This approach has proven itself particularly useful in cases when one wants to study a gene that is essential for cell growth​32,33​. These CRISPR-based techniques have all been successfully implemented to expand the genetic toolkit available for genetic perturbation and analysis in otherwise unwieldy microbial organisms. Genomic perturbations using CRISPR technologies have greatly facilitated functional genomic analysis in ​Mycobacterium tuberculosis ​— a notoriously difficult-to-manipulate pathogen and the causative agent of tuberculosis. CRISPRi techniques have bypassed the need for homologous recombination and allowed precise knock-down expression of ​M. tuberculosis​ genes​34–36​. The ability to genetically regulate genes to confirm their essentiality and study their function has important applications, but has been difficult in ​Mycobacteria​ with traditional methods, where much of the genome remains uncharacterized. By optimizing a novel CRISPRi platform in ​Mycobacteria​, the necessity of several putative essential genes, including those involved in folate metabolism was readily determined​37​ (Figure 1b). Given the scalable nature of this platform, future high-throughput CRISPRi repression studies