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.

Genome editing in cancer:Challenges and potential opportunities

Ever since its mechanism was discovered back in 2012,the CRISPR/Cas9 system have revolutionized the field of genome editing.While at first it was seen as a therapeutic tool mostly relevant for curing genetic diseases,it has been recently shown to also hold the potential to become a clinically relevant therapy for cancer.However,there are multiple challenges that must be addressed prior to clinical testing.Predominantly,the safety of the system when used for in-vivo therapies,including off-target activity and the effects of the double strand break induction on genomic stability.Here,we will focus on the inherent challenges in the CRISPR/Cas9 system and discuss various opportunities to overcoming these challenges.