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).

Indiscriminate ssDNA cleavage activity of CRISPR-Cas12a induces no detectable off-target effects in mouse embryos

Dear Editor,Newly discovered characteristics like"collateral effect"or trans-cleavage in CRISPR-Cas13 and CRISPR-Cas12 systems have enabled their usage in nucleic acid detection(Gootenberg et al.2017,2018;Chen et al.2018).The collateral RNA cleavage of Cas13a has been reported to be harmful for cell development(Wang et al.2019;Buchman et al.2020).As a representative gene editor of CRISPR-Cas12 system,CRISPR-Cas12a(Cpf1)holds great potential for therapeutic applications in the future(Zetsche et al.2015;Koo et al.2018;Campa et al.2019).

Precise genome editing without exogenous donor DNA via retron editing system in human cells

Dear Editor,CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair(HDR),albeit with relative low efficiency due to the inefficient delivery of exogenous DNA(Cox et al.,2015;Gao,2021).Retrons are bacterial phage-defense related operons composed of a specialized reverse transcriptase(RT)and a relevant non-coding RNA(ncRNA)which can be partially reverse tran-scribed by RT initiating at a conserved guanosine(G)residue to produce a multicopy single-stranded DNA(msDNA)(Yee et al.,1984;Millman et al.,2020).After being reverse transcribed,the msDNA is usually covalently teth-ered to the ncRNA through the 2',5'-phosphodiesterbond between the priming G in ncRNA and 5'end of msDNA(Dhundale et al.,1987).The reverse transcription process,of which the specialized RT recognizes the unique secondary structure of retron ncRNA,is highly specific(Hsu et al.,1989).Additionally,desired msDNA can be generated in vivo by replacing the dispensable region of retron ncRNA with desired sequences(Mirochnitchenko et al.,1994;Simon et al.,2019).Therefore,retrons are promising biological sources for in vivo generation of DNA donors for HDR-me-diated precise genome editing.

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.