Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

Dear Editor,RNA knockdown in vivo carries significant potential for dis-ease modeling and therapies.Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes,strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of genome DNA is not appropriate,and RNA virus infection diseases when pathogenic DNA is not available(such as SARS-Cov-2 and MERS infections).

Inflammation-induced inhibition of chaperone-mediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells

Macroautophagy has been implicated in modulating the therapeutic function of mesenchymal stromal cells(MSCs).However,the biological function of chaperone-mediated autophagy(CMA)in MSCs remains elusive.Here,we found that CMA was inhibited in MSCs in response to the proinflammatory cytokines interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α).In addition,suppression of CMA by knocking down the CMA-related lysosomal receptor lysosomal-associated membrane protein 2(LAMP-2A)in MSCs significantly enhanced the immunosuppressive effect of MSCs on T cell proliferation,and as expected,LAMP-2A overexpression in MSCs exerted the opposite effect on T cell proliferation.This effect of CMA on the immunosuppressive function of MSCs was attributed to its negative regulation of the expression of chemokine C-X-C motif ligand 10(CXCL10),which recruits inflammatory cells,especially T cells,to MSCs,and inducible nitric oxide synthase(iNOS),which leads to the subsequent inhibition of T cell proliferation via nitric oxide(NO).Mechanistically,CMA inhibition dramatically promoted IFN-γplus TNF-α-induced activation of NF-κB and STAT1,leading to the enhanced expression of CXCL10 and iNOS in MSCs.Furthermore,we found that IFN-γplus TNF-α-induced AKT activation contributed to CMA inhibition in MSCs.More interestingly,CMA-deficient MSCs exhibited improved therapeutic efficacy in inflammatory liver injury.Taken together,our findings established CMA inhibition as a critical contributor to the immunosuppressive function of MSCs induced by inflammatory cytokines nd highlighted a previously unknown function of CMA.

An engineered xCas12i with high activity,high specificity,and broad PAM range

Dear Editor,The clustered regularly interspaced short palindromic repeats-Cas(CRISPR-Cas)systems,including type II Cas9 and type V Cas12 systems,which serve in the adaptive immunity of prokaryotes against viruses,have been developed into genome-editing tools(Anzalone et al.,2020;Doudna,2020).Compared with type II systems,the type V systems including V-A to V-K showed more functional diversity(Yan et al.,2019).Amongst them,Cas12i has a relatively smaller size(1,033-1,093 aa),compared to SpCas9 and Cas12a,and has a 5'-TTN protospacer adjacent motif(PAM)preference(Yan et al.,2019).

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.