The Type Ⅰ-A CRISPR-Cas Complex Purification From Sulfolobus Islandicus REY15A

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats and their associated genes)is an adaptive immune system used by prokaryotes to defend against foreign nucleic acids,which is distributed in approximately 40% of bacteria and 90% of archaea.Now,The CRISPR-Cas system is divided into two Class(Class1 and 2)and six Types(Type I-Ⅵ).The Cas9 and Cas12 systems have been widely used for gene editing in a variety of different eukaryotes and prokaryotes,leading to a great revolution in biology.For this reason,the 2020 Nobel Prize in Chemistry has also been awarded to this revolutionary technology.However,the Cas9 and Cas12 systems are not widely distributed in prokaryotes,and the large number of CRISPR immune systems in archaea and bacteria belong to Type I and Type III CRISPR system of Class1.The three CRISPR systems found in the Sulfolobus islandicus REY15 A,a model organism for archaea research,are Type I-A,Cmr-ɑ and Cmr-β.Over the past 10 years,our research team by using genetics,biochemistry and structural biology methods for two kinds of type III-B CRISPR immune system,the Cmr-ɑ and Cmr-β,have studied systematically.However,for the I-A system that showed the highest activity in the gene editing of the S.islandicus REY15 A,remained at the preliminary genetic characterization of its immune activity.The main reason is that the purification of I-A CRISPR complex is difficult to achieve.So the main purpose of this study is through different methods to get an activity Type I-A complex.It will provide a solid foundation for the further development and utilization of the immune system,as well as the study of the interaction and molecular ecology between microorganisms and their viruses.In this study,the optimal purification of I-A complex was purified through the extensive screening of eight different expression plasmids in the early stage.And,the best method for the purification of I-A complex from Sulfolobus islandicus REY15 A was found.In order to facilitate the subsequent protein purification and biochemical experiments,we used endogenous CRISPR system to perform the following operations on the I-A CRISPR system gene cluster :(1)The promoter of the I-A CRISPR system gene cluster was replaced to increase the expression of the complex.(2)The 5’ terminal of csa5 gene on the genome was added with 10×His tag to facilitate the subsequent protein purification.(3)Delete the original CRISPR Array on the genome for the convenience of subsequent biochemical experiments.Promoter replacement and genomic tagging purification methods effectively increased the amount of protein available.However,through the subsequent detection of the obtained protein,we found that the harvested protein only contained Cas7,Cas5,Cas8,Csa5.The backbone did not contain Cas3’and Cas3",which was similar to the purification situation in Type I-D,Type I-E and Type I-F.However,this is contrary to the experimental conclusion that Plagens assembled Type I-A complex of Thermoproteus Tenax in vitro,that is,Cas3’and Cas3 "are necessary parts of the complex composition and play an important role in the stability of the complex.However,our subsequent purification experiments in the expression plasmid p Se SDN10His-Cas5-Cas3’-C6 His showed that the Cas3’protein did not bind tightly to the complex backbone and was more likely to be recruited in the later stage by the complex backbone.Then,in order to verify the in vitro activity of the I-A complex,we purified Cas3’and Cas3" proteins separately from the archaea,and tried to co-incubate them with the existing complex backbone,hoping to see the cleavage activity in vitro.In this study,a relatively complete complex backbone was purified from the host for the first time.Although there was no expected cleavage activity in vitro,it is a good foundation for future experiments.Moreover,there were many phenomena in the purification process that were different from those published in previous papers,which were worthy of further detailed studies.