A compact Cascade–Cas3 system for targeted genome engineering

Abstract

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions.Here, we utilize a Cas3-based system featuring a processive nuclease for genome engineering. This minimal Cascade-Cas3 system (Type I-C), programmed with a single crRNA, was optimized to generate deletions with near-100% efficiency, and used to rapidly generate large deletions ranging from 7 -424 kb in Pseudomonas aeruginosa. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were highly variable, but successfully specified by a homology-directed repair (HDR) template. HDR was much more efficient when lesions were generated by Cas3, compared to Cas9. The minimal Type I-C system

Structural basis for assembly of non-canonical small subunits into type I-C Cascade

“…2f). Taken together, our model provides functional insights into one of the most prevalent CRISPR-Cas systems in bacteria which may serve as a blueprint for developing a minimal Cascade for genome editing 24,25 .…”

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

“…In another attempt to construct ΔpqsA in PAO1 λIF (data not shown), we identified false positive clones carrying mutated editing plasmid in which the spacer and the downstream repeat sequence was excised ( Supplementary Figure S10), suggesting mutations in cas genes or the self-targeting mini-CRISPR in the editing plasmid are the major reasons for the emergence of false positive clones (49). Hence, future efforts to increase the editing efficiencies should focus on preventing these spontaneous mutations, such as modifying the repeat sequence in the mini-CRISPR to prevent the spacer excision via homologous recombination between the two repeats (15).…”

“…An alternative strategy to overcome Acrs is to overexpress the anti-CRISPR-associated gene ( aca ) which represses acr and in turn actives CRISPR-Cas systems (50). This strategy has been demonstrated to be effective in a type I-C Cascade-mediated genome editing in a PAO1 variant which is lysogenized by a recombinant DMS3m phage expressing the anti-CRISPR gene acrIC1 (15). However, robustness of Aca-mediated anti-anti-CRISPR strategy might be compromised in clinical strains owing to their complicated genetic backgrounds.…”

“…Employing the strategy, editing of several genetically recalcitrant organisms such as the industrial bacterium Clostridium pasteurianum ATCC6013 (type I-B) (12), the multidrug resistant Pseudomonas aeruginosa genotype PA154197 (type I-F) (13), and a human microbiome Lactobacillus crispatus NCK1350 (Type I-E) (14) has been achieved. Furthermore, the processive DNA degradation fashion of the type I signature nuclease Cas3 has enabled long-range genomic deletions in both bacteria (employing the Type I-C Cascade from P. aeruginosa ) (15) and human embryonic stem cells (employing the Type I-E Cascade from T. fusca ) (16) which are inaccessible by the Class 2 effectors. Recently, the type I-B, type I-E, and type I-F systems have also been repurposed for transcription modulation in human cells (17,18).…”

Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application

“…Therefore, we can reuse the endogenous type I CRISPR-Cas system in bacteria and archaea by synthesizing a mini CRISPR array. This will allow the endogenous Cascade-Cas3 complex to be redirected to the genome and reused for targeted killing, genome editing, or transcriptional control (Csorgo et al, 2020). In recent years, reusing these extensive and endogenously encoded CRISPR-Cas systems for "built-in" genome editing has become a simple, efficient and promising genetic manipulation strategy in prokaryotes (Cheng et al, 2017;Zhang et al, 2018).…”