Academic journal article Genetics

Advances in Engineering the Fly Genome with the CRISPR-Cas System

Academic journal article Genetics

Advances in Engineering the Fly Genome with the CRISPR-Cas System

Article excerpt

THROUGH more than a century of study and extensive development of genetic tools, Drosophila melanogaster has become a premier system for understanding complex biological processes at the molecular, cellular, and organismal levels. Despite this strength, until recently, making precise modifications to the genome was challenging, labor intensive, and had a low frequency of success. The first successful genome-editing strategies induced homologous recombination through P-element excision, or the in vivo generation of linear templates using multiple transgenic constructs (Gloor et al. 1991; Banga and Boyd 1992; Rong and Golic 2000; Gong and Golic 2003; Huang et al. 2008, 2009). More recent strategies have relied on the generation of a targeted doublestrand break (DSB) in the genome to trigger DNA repair by the cellular repair machinery-a process that can be co-opted to precisely modify genomic sequences. Targeted DSBs were first generated by programmable nucleases, either zinc-finger nucleases (ZFNs; Bibikova et al. 2002) or transcription activator-like effector nucleases (TALENs; Liu et al. 2012). The more recent co-option of highly programmable bacterial adaptive immune systems for generating targeted DSBs has resulted in an unprecedented level of control of the genome of nearly any organism (Harrison et al. 2014). With this simple, two-component, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, the researcher need only provide a small targeting RNA, which is easily synthesized, and the bacterial nuclease. Because all of these genome-editing strategies rely upon the cellular DNA-repair machinery, lessons learned from earlier approaches have driven the rapid advance of CRISPR-based approaches.

CRISPR-Cas Systems in Bacterial Immunity

The first CRISPR locus was identified in 1987 based on its highly repetitive sequence (Ishino et al. 1987), but it took nearly 20 years until a definitive link was made between these repeats and a role in adaptive immunity (Barrangou et al. 2007). Impressively, it was only 6 years after this seminal discovery that multiple groups published successful co-option of the system for genome editing (Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013b). In the short time since the original demonstrations of genome editing using the bacterial CRISPR-Cas system, it has rapidly become a nearly universal tool in biological research. The continued development of this system builds on understanding its fundamental role in bacterial and archaeal immunity.

The CRISPR locus is an array of alternating repeats and spacer sequences that essentially provides a chronological history of the viruses and plasmids that have invaded a given bacterial strain (Barrangou etal. 2007). CRISPR-based immunity involves three steps that are all critical for protecting the bacteria from invading viruses: adaptation, expression, and interference (van der Oost et al. 2009; Makarova et al. 2011). In the adaptation step, fragments of foreign DNA are incorporated into the CRISPR locus as new spacers. During expression, the transcription and subsequent processing of the CRISPR locus provides an RNA template for recognition of complementary protospacer sequences in the invading DNA. In the final interference step, the invading DNA is cleaved and inactivated by an RNA-guided Cas effector protein. To allow the immune system to distinguish invader DNA from genomic DNA incorporated in the CRISPR array, stable binding by the effector protein and subsequent DNA cleavage requires a protospacer adjacent motif (PAM) present only in the targeted DNA.

The constantly active arms race between virus and host has resulted in a large amount of variation in CRISPR-Cas systems, which are present in most archaea and about half of all bacteria and currently divided into two classes, six types, and numerous subtypes based on the complement of Cas genes associated with the CRISPR locus (Makarova et al. 2011, 2015; Shmakov et al. …

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