Versatile and efficient mammalian genome editing with Type Ⅰ-C CRISPR System of Desulfovibrio vulgaris

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins.While type I CRISPR systems in Class I may offer greater specificity and versatility,they are not well-developed for genome editing.Here,we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris(Dvu)for efficient and precise genome editing in mammalian cells and animals.We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy.These edited pig cells can serve as donors for generating transgenic cloned piglets.The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair.Additionally,we adapted the Dvu-Cascade effector for cytosine and adenine base editing,developing Dvu-CBE and Dvu-ABE systems.These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks.Off-target analysis confirmed the high specificity of the Dvu type I-C editor.Our findings demonstrate the Dvu type I-C editor′s potential for diverse mammalian genome editing applications,including deletions,fragment replacement,and base editing,with high efficiency and specificity for biomedicine and agriculture.

The influence of the copy number of invader on the fate of bacterial host cells in the antiviral defense by CRISPR-Cas10 DNases

Type Ⅲ CRISPR-Cas10 systems employ multiple immune activities to defend their hosts against invasion from mobile genetic elements(MGEs),including DNase and cyclic oligoadenylates(cOA)synthesis both of which are hosted by the type-specific protein Cas10.Extensive investigations conducted for the activation of Cas accessory proteins by cOAs have revealed their functions in the type Ⅲ immunity,but the function of the Cas10 DNase in the same process remains elusive.Here,Lactobacillus delbrueckii subsp.Bulgaricus type Ⅲ-A(Ld)Csm system,a type Ⅲ CRISPR system that solely relies on its Cas10 DNase for providing immunity,was employed as a model to investigate the DNase function.Interference assay was conducted in Escherichia coli using two plasmids:pCas carrying the LdCsm system and pTarget producing target RNAs.The former functioned as a de facto“CRISPR host element”while the latter,mimicking an invading MGE.We found that,upon induction of immune responses,the fate of each genetic element was determined by their copy numbers:plasmid of a low copy number was selectively eliminated from the E.coli cells regardless whether it represents a de facto CRISPR host or an invader.Together,we reveal,for the first time,that the immune mechanisms of Cas10 DNases are of two folds:the DNase activity is capable of removing low-copy invaders from infected cells,but it also leads to abortive infection when the invader copy number is high.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genomewide RNA Interference Screening

Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single-and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Visualization of phage DNA degradation b.y a type I CRISPR-Cas system at the single-cell level

Background： The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level. Methods： In this study, we fluorescently label phage lambda DNA in vivo, and track the labeled DNA over time to characterize DNA degradation at the single-cell level. Results： At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our antilambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence ofinvasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell. Conclusions： With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.