Optimization Of Cytosine Base Editor Based On CRISPR/Cpf1 System

Recently,base editors have become an integral part of gene editing technology.It has many advantages,such as no double-strand breaks,no exogenous donor template,and editing in non-dividing cells.The base editor has been used in point mutation disease animal model construction,crop trait improvement,disease gene therapy.The most widely used base editor is developed based on CRISPR/Cas9 system.The most commonly used base editor is developed based on Sp Cas9 system.It inherits the characteristic of Sp Cas9 to recognize G/C-rich PAM(NGG),which limits its targetable scope.Subsequently,a base editor based on CRISPR/Cpf1 system was also developed,which has the characteristic of recognizing T/A-rich PAM.It is formed by fusing r APOBEC with nuclease-inactivated d Lb Cpf1 and named d Lb Cpf1-BE0.However,there are some shortcomings,such as low editing efficiency,motif preference.These shortcomings hinder the development and application of base editors based on the CRISPR/Cpf1 system.In view of the low efficiency of existing Cpf1-based base editing tools,we used different strategies to improve editing efficiency in this study.First,we use h APOBEC3 A to construct d Lb Cpf1-h A3 A,which has higher cytosine deaminase activity,and the editing efficiency can reach up to 50%.For some d Lb Cpf1-BE0 incompetent sites,the editing efficiency of d Lb Cpf1-h A3 A can also reach 20%.Compared with d Lb Cpf1-BE0,the average editing efficiency of d Lb Cpf1-h A3 A has been significantly improved.Due to the use of h APOBEC3 A,the active window of d Lb Cpf1-h A3 A has also been widened to C6-C13,which will also lead to the occurrence of bystander effects.We use h A3A-Y130 F and h A3A-Y132 D to build base editors,which can narrow the active window while maintaining editing efficiency.We also used another variant h A3A-N57 G to construct d Lb Cpf1-h A3A-N57 G that specifically deaminates C in the TCR(R=A/G)motif.This editor can greatly reduce the bystander effect while maintaining higher accuracy.In addition to the above features,our constructed d Lb Cpf1-h A3 A and its variants can efficiently edit all motifs,which is different from d Lb Cpf1-BE0.Therefore,we successfully constructed an efficient base editor based on CRISPR/Cpf1 system.In addition to replacing deaminase,we also focus on the open state of chromatin to further improve the efficiency of the base editor based on CRISPR/Cpf1 system.We use d Cas9/sg RNA binding on both sides of the target site to open the chromatin structure locally,thereby increasing the efficiency of targeted editing.But for different sites,the efficiency improvement is different,and the maximum can be doubled.We also analyzed the Cpf1-independent off-target and found that the off-target using a shorter(14-15nt)dead sg RNA was almost undetectable.In summary,we optimized the cytosine base editor based on CRISPR/Cpf1 system to achieve efficient base editing and expand the base editing toolbox.In this study,a series of base editors constructed using h APOBEC3 A and its variants have different characteristics and provide more choices for future applications.In addition,the strategy of combining d Cas9 with adjacent positions can further improve editing efficiency and promote the application and development of CRISPR/Cpf1 based base editing tools.