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CRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a’s target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central ‘binding seed’ where perfect base pairing is absolutely required for target binding, and a separate ‘nuclease switch’ where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a’s RNA binding and cleavage behavior for RNA-targeting tool development.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system and has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities, which was first identified in Leptotrichia shahii CRISPR-Cas13a. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a and other CRISPR-Cas systems. Various types of CRISPR-Cas systems, I-B, II-C, III-A, III-D, III-like and VI-A, were unevenly distributed among the Leptotrichia genomes, accounting for 10 (34.4%), 1 (2.6%), 6 (15.4%), 6 (15.4%), 3 (7.7%) and 11 (37.9%) of the 29 strains, respectively, while 8 (20.5%) strains had no CRISPR-Cas system. The Cas13a effectors were highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii and their collateral ssRNA cleavage activities, resulting in cytotoxicity to host cell, were found to be maintained. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 2 (0.5%) of 400 spacers through CRIPSR-Cas I-VI were shared by two strains. The organisation and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Genome engineering by site-specific nucleases enables reverse genetics and targeted editing of genomes in an efficacious manner. Contemporary revolutionized progress in targeted-genome engineering technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related RNA-guided endonucleases facilitate coherent interrogation of crop genome function. Evolved as an innate component of the adaptive immune response in bacterial and archaeal systems, CRISPR/Cas system is now identified as a versatile molecular tool that ensures specific and targeted genome modification in plants. Applications of this genome redaction tool-kit include somatic genome editing, rectification of genetic disorders or gene therapy, treatment of infectious diseases, generation of animal models, and crop improvement. We review the utilization of these synthetic nucleases as precision, targeted-genome editing platforms with the inherent potential to accentuate basic science “strengths and shortcomings” of gene function, complement plant breeding techniques for crop improvement, and charter a knowledge base for effective use of editing technology for ever-increasing agricultural demands. Furthermore, the emerging importance of Cpf1, Cas9 nickase, C2c2, as well as other innovative candidates that may prove more effective in driving novel applications in crops are also discussed. The mined data has been prepared as a library and opened for public use at www.lipre.org.

Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012–2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.