Construction Of Genome Edited Rabbit Model Using SpG And SpRY Systems With Broadened PAM Compatibility

CRISPR/Cas9 gene editing system is originated from the adaptive immune system of bacteria and archaea,under the guidance of a sg RNA,the system can achieve highly efficient sequence-specific targeting.However,the innate mechanism for Cas9 to avoid autoimmuity,protospacer adjacent motif(PAM)recognition,largely limited the targeting scope of CRISPR/Cas9 systems.In 2020,two SpCas9 variants,SpG and SpRY,were constructed via structureguided engineering,these variants had significantly improved the targeting scope of the CRISPR/Cas9 system by reducing their PAM preference.SpG can efficiently recognize NGN PAM,which accounts for 20.45% of human genome;SpRY is “near-PAM less”,which can efficiently recognize NRN PAM(R=A or G)that accounts for 50.01% of the genome,and has considerable recognition efficiency for part of the NYN PAMs(Y=C or T)that accounts for 49.99% of the genome.The construction of base editors(BEs)that based on SpG and SpRY can effectively target sites with non-canonical PAMs,which is unaccessible for previous editing systems.However,the editing efficiency of these two systems have only been tested in cell lines,plants,and zebrafish,and were not yet applied in constructing genome-edited mammals.Rabbit is a classic mammalian model,construction of genome edited rabbit disease models has great value for practical biomedical research.Previously,the application of BE systems in rabbits has successfully simulated many human pathogenic point mutations.However,the strict PAM preference of the SpCas9 makes target many sites inaccessible for canonical BEs.Notably,the novel SpG and SpRY have great potentiality to solve this problem.First,6 sites with NGN PAM in rabbit embryos were selected to estimate the editing efficiency of the SpG system.Both SpG-BE4 max and SpG-ABEmax were tested,and NG-Cas9,an early Cas9 variant that recognizes NGN PAM,was applied as a control.For editing at the Tyr-p.R285 X,Dmd-p.Q785 X,and Mstn-p.C313 Y loci,SpGBE4max-mediated C→T editing efficiency ranged from 50% to 87.5%,with average editing frequencies of 43.4% to 91.1%,the editing activity of SpG-BE4 max is significantly better than that of NG-BE4max(editing efficiency 30%～50%,average mutation frequency 8.0%～76.0%).For editing at the Sod1-p.I151 T and Lmna-p.L530 P loci,SpG-ABEmax-mediated A→G base editing efficiency was 36.4%～92.3%,with average editing frequencies of 70.8%～90.9%,the editing activity of SpG-ABEmax is significantly better than that of NG-ABEmax(can only target Sod1-p.I151 T loci with editing efficiency of 44.4%,and 56.5% average mutation frequency,no efficiency at Lmna-p.L530 P loci).Based on the validation of embryonic editing efficiency,this study further investigated the efficiency and accuracy of SpG in model construction by using SpGABEmax to construct an amyotrophic lateral sclerosis(ALS)rabbit disease model with the Sod1-p.I151 T mutation.All F0 individuals carried the desired mutation,and the mutation frequency ranges from 81.0% to 97.0%,no unwanted mutations were detected.Gait analysis,electromyography,and histological studies proved that the Sod1-p.I151 T mutant ALS rabbit model has shortened stride length,fibrillation,abnormal motor unit potentials,muscle atrophy,and neuronal death.Suggesting that the rabbit ALS model exhibits motor dysfunction,denervation and reinnervation,muscle atrophy,and neurodegeneration,which were consistent with typical clinical characteristics of human ALS patients.Base on the rabbit model,the study further elucidated the molecular pathological mechanism of Sod1-p.I151 T mutation mediated ALS,the p.I151 T mutation of SOD1 can lead cell apoptosis by inducing chronic endoplasmic reticulum stress.Although SpG has a higher targeting scope than the original SpCas9,it still cannot recognize non-canoncial PAMs such as NCN,NTN,and NAN.The SpRY system was developed based on the SpG system with further expanded PAM compatibility.In this study,the editing efficiency of SpRY system at non-classical PAM sites were confirmed in rabbit embryos.Several target site with non-classical PAM were selected for analyzing the editing activity of SpRY-BE4 max and SpRY-ABEmax in rabbit embryos.For editing at Arpp21-p.P529 L,Kcnj11-p.F333 F,Kcnj11-p.H186 H,and Gck-p.R186 X loci,SpRY-BE4max-mediated C→T editing efficiency ranged from 66.7% to 100%,with average editing frequencies of 52.5% to 89.0%.For editing at Anxa11-p.D40 G,Kcnj11-p.Y335 C,Kcnj11-p.H259 R and Kcnj11-p.I296 V loci,SpRY-ABEmax-mediated A→G base editing efficiency was 50.0%-80.0%,with average editing frequencies of31.4%～94.1%.SpRY-BE4 max and SpRY-ABEmax has efficient targeting activity at non-canonical PAM sites.To further validate the effectiveness of the SpRY system in constructing rabbit model,co-targeting of two non-canonical PAM site,Kcnj11-p.H259 R and Kcnj11-p.I296 V,were induced via the application of SpRY-ABEmax to generate a congenital hyperinsulinemia(CHI)rabbit model.All F0 generation rabbits carried both target mutations,the mutation frequencies ranges from 42.0% to 82.0% at Kcnj11-p.H259 R loci and 46.0% to 97.0% at Kcnj11-p.I296 V loci,respectively.Genotyping showed that SpRY-ABEmax did not cause any unwanted mutations.The CHI rabbit model exhibited typical clinical symptoms of human CHI,including decreased fasting blood glucose,increased serum insulin levels,and pancreatic insulin deposition.The modle rabbite accurately recapitulated human CHI caused by functional loss-of-function mutations of KCNJ11 gene.In summary,this study confirmed the editing efficiency of the SpG system on NGN PAM sites in rabbit embryos,and proved its superior targeting efficiency over the original NG-Cas9 system,and a genome edited rabbit ALS modle was constructed by using SpG system for the first time.This is one of the earliest mammalian models constructed by SpG system,and it is also the first reported genome edited rabbit model for human neurodegenerative disease.The model successful recapitulated typical clinical symptoms of human ALS and completely mimicked the full course of ALS pathogenesis,exhibiting better accuracy than traditional drug induction model.Such model can be ideal platform for future researches of disease pathological mechanisms and preclinical tests for diagnostic and therapeutic approaches.In addition,this study further verified the editing efficiency of the “near-PAM less” Cas9 variant,SpRY,in rabbit embryos,and constructed CHI model rabbits by using SpRY for the first time.The application of SpG and SpRY systems has greatly broadened the targeting scope for constructing genome edited rabbits,and provided theoretical and technical support for future large-scale construction of rabbit models for human diseases.