Template-free Repair Outcome Profiling And Editing Activity Optimization Of RISPR/Cas12a System In A High-throughput Manner

CRISPR/Cas systems are adaptive immune systems found in bacteria and archaea to defend prokaryotes against bacteriophage and other mobile genetic elements,which are deemed as revolutionary tools for genetic engineering.Two Class II CRISPR/Cas systems,Type II Cas9 and Type V-A Cas12 a,utilizing single protein effectors combined with guide RNAs to cleavage complementary DNA targets,are widely used in various species and fields.Compared with Cas9,Cas12 a system exhibits various distinct properties: having more compact structure,possessing both DNase and RNase activity,requiring a single short crRNA,recognizing targets with T-rich PAMs,and generating PAM-distal staggered ends as well as low off-target effects.Since CRISPR/Cas12 a was developed in 2015,over 10 Cas12 a orthologues were identified and demonstrated to possess gene editing activity in mammalian cells.However,the mutational profiles and editing efficiency of diverse Cas12 a orthologues have not been systematically investigated.Study on these issues will provide theoretical guidance for explaining the mechanism of Cas12 a system and reducing off-target effect,which is also valuable for engineering new Cas12a-based gene editing tools in various fields.In this work,by constructing genome-integrated self-cleaving paired crRNA-target libraries,we used a high-throughput approach to investigate template-free repair outcome profiles and editing efficiency of CRISPR/Cas12 a system.Four achievements are shown as follows:(1)We reported detailed profiling of the template-free repair outcome of the Cas12 a system in mammalian cells.Major reproducible mutation types generated by Cas12 a are deletions,consisting of NHEJ(Non-homologous end joining)-mediated short deletions(1～8bp)and MMEJ(Microhomology-mediated end joining)-mediated longer deletions(12～18bp),which are different from those by Cas9.Meanwhile,we identified three factors influencing the profile of editing outcomes: genetic backgrounds(DNA repair pathway preference),local sequence context flanking target sites and alternative CRISPR/Cas effectors.(2)We comparatively studied editing activity of ten Cas12 a orthologues in HEK293 T and RKO cells.Three Cas12 a candidates(Bs Cas12 a,Pr Cas12 a,and Mb3Cas12a)exhibited promising potential owing to their having more compact structures and comparable editing efficiency to well-characterized As Cas12 a and Lb Cas12 a.(3)We generated 3-Arginine substitution variants(3Rv)of 3 Cas12 a orthologues,including Bs Cas12a-3Rv(K155R/N512R/K518R),Pr Cas12a-3Rv(E162R/N519R/K525R),and Mb3Cas12a-3Rv(D180R/N581R/K587R)via a structure-guided protein engineering approach.These orthologues demonstrated a wider PAM recognition range(NTTV,NTCV,TRTV)and higher editing activity.Then we also characterized and compared PAM recognition preferences among 3 Cas12 a variants:At R(-3)(R = A,G)in targets with PAM TRTV: PrCas12a-3Rv and Bs Cas12a-3Rv notably preferred G over A;while Mb3Cas12a-3Rv exhibited less bias for base A and G.At N(-4)(N = A,C,G,T)in targets with PAM NTCV: Pr Cas12a-3Rv and Bs Cas12a-3Rv notably preferred T over V(V =A,C,G);while Mb3Cas12a-3Rv behaved differently,which preferred A comparable to T and exhibited weaker recognition of base C and G.(4)We systematically studied the effect of crRNA folding structure on Cas12 a editing activity.Two categories of crRNA folding structure were identified detrimental to Cas12 a editing activity: Secondary structure formed intra-spacer and unexpected base pairing with direct repeat(5’handle of crRNA).We suggested that shorter spacer length or alternative loop region sequences in crRNA 5’scaffold would improve the editing performance.In conclusion,we used a high-throughput approach to systematically profile template-free repair outcome of CRISPR/Cas12 a and comparatively evaluate editing activity of 10 Cas12 a orthologues.We identified three potential Cas12 a candidates in mammalian cells.Through structure-guided engineering and ameliorated crRNA designing,we achieved to optimize Cas12 a system with an expanded target range,improved editing activity and predictable major repair products.These optimization and improvement will facilitate development of diverse Cas12a-based tools for gene editing,transcriptional regulation,live imaging and lineage tracing in mammalian cells and various species,and also contribute crop breeding,microbial metabolic engineering and clinical applications.