Application Of A Single SgRNA-Mediated Programmed Enzyme CRISPR-Cpf1 As Artificial Restriction Enzyme In DNA Assembly

The CRISPR-Cas system is a programmable immunologically adaptive(acquired)immune system in prokaryotes.Exogenous genes are stored as spacers and inserted into CRISPR arrays,followed by RNA-directed nuclease effectors to mediate the DNA damage of any invasive mobile genetic element.The CRISPR-Cas system is divided into two categories based on protein effectors.Type 1 is a multi-subunit effector complex and class 2 is a single protein effector.Based on different families of effector proteins,category 2 systems are further divided into categories type II,V,VI and nine subtypes.Where type II and type V CRISPR-effector nucleases Cas9 and Cpfl are "universal" DNA endonucleases that can be programmed through the appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at the preselected position(consisting only of short So-called PAM limit).The CRISPR-Cas system can therefore be used as a virus defense mechanism and can also be used to reduce the spread of fluid genetic factors.The immune system has become a powerful genomic editing tool in the past few years and has become a molecular toolkit for a wide range of organisms.Cpfl,a novel class 2 family of CRISPR-Cas RNA-programmable endonucleases,also known as Cas12a,has the unique feature of single RNA-guided endonuclease lacking tracrRNA,utilizing the T-rich PAM recognizes and cleaves double-stranded DNA targets that are complementary to the crRNA guide and away from the PAM,resulting in an interleaved DNA duplex that sticks 5nt of sticky ends.This study is based on the programmable endonuclease Cpfl gene can be edited for the background,the use of Cpfl is derived from Francisella tularensis novicida U112 strain(FnCpfl),the gene sequence number:MF193599,encoding gene sequence length 3903 bp,encoding 1301 amino acids.We successfully constructed the heterologous expression plasmid of FnCpfl enzyme fused to MBP tag,and realized its highly secretive expression and high enzyme activity in the expression system of Escherichia coli BL21(DE3).In addition,we not only successfully transcribed single-strand guide RNA in vitro,but also established a set of efficient FnCpfl enzyme digestion system.Through a series of bold assumptions and experiments,we have successfully completed the application of programmable enzyme CRISPR-FnCpf1 mediated by single sgRNA as an artificial restriction endonuclease,target fragments transfer in plasmids and multi gene assembly.Compared with the traditional type ? restriction endonucleases,the method has more advantages,such as prominent sticky ends longer without the need to be limited by internal restriction sites and the sequence itself within the target fragment and can replace multiple type ? restriction endonucleases,Low cost,ease of use,etc.,making it a very attractive alternative or supplement for genomic engineering.The assembly of large-scale constructs from small fragments of DNA in recent years has been of great significance and become a key technology in the field of synthetic biology.Although there are many kinds of gene assembly methods available,different assembly techniques have different applicability and limitations.The usual strategy is the joint use of several technologies.In this study,a new method for in vitro DNA assembly mediated by the programmable endonuclease CRISPR/Cpf1 can be used as a powerful complement to traditional gene cloning methods.Using FnCpfl enzyme digestion successfully completed the target fragment in the transfer between plasmids and other experiments;then,this experiment also pioneered the concept of"degenerate sgRNA" and completed the feasibility test,found that degenerate sgRNA guided FnCpf1 can also perform restriction enzyme digestion of target DNA;Finally,we boldly proposed using the end of 6nt mismatched.sgRNA with the target DNA to guide the FnCpf1 enzyme cleavage,fortunately found that the end of 6nt mismatched sgRNA-guided FnCpfl also has a significant cleavage effect.Such kind of method showed unexpected superiority,which can be readily harnessed instead of restriction enzymes for routine use in molecular cloning and also makes seamless assembly more concise in vitro.More importantly,the use of the heterologous expression programmable enzyme FnCpf1,which is only directed by a single sgRNA,in combination with the T5 exonuclease greatly improves the efficiency of multisegment assembly.