CRISPRClusteredRegularlyInterspacedShortPalindromicRepeats (clustered, regularly spaced, short palindromes, repetitive sequences), composed of palindromes repeats and their spacer sequences, is an acquired immune method among most bacteria and archaea. There are also some CRISPR-associated genes near the CRISPR characteristic sequence, encoding a series of Cas proteins, collectively called the CRISPR-Cas system.
interval sequence comes from foreign invading DNA. As a fingerprint to identify the identity of foreign invaders, it corresponds to the original interval sequence (protospacer) on the invading DNA. As the original interval sequence for identification, it is characterized by the proximity sequence extending at both ends. It is very conservative and is called the proximity motif of the original interval sequence (protospacer adjacentmotif,PAM).
virus (phage) and plasmid invade the cell for the first time, the proteins encoded by Cas1 and Cas2 will scan the foreign DNA and identify the conserved PAM region, and then the non-conserved DNA sequence adjacent to PAM will be taken as the candidate original spacer sequence. Subsequently, the Cas1/2 protein complex cuts the original spacer sequence from the foreign DNA, and inserts the original spacer sequence downstream near the lead region of the CRISPR sequence with the assistance of other enzymes. Then, the DNA will repair and close the open double-stranded gap. In this way, a new interval sequence is added to the CRISPR sequence of the genome, forming an immune "memory" of viral DNA ".
When foreign DNA invades again, the CRISPR/Cas system will accurately strike the foreign DNA according to the immune "memory" formed by the first invasion. This process is carried out in two steps-crRNA synthesis and crRNA-guided RNA binding and shearing.
CRISPR region. This sequence acts as a promoter to initiate the transcription of subsequent CRISPR sequences and transcribes into two RNAs, pre-CRISPR-derived RNA(pre-crRNA) and trans-acting crRNA(tracrRNA). Among them, the tracrRNA is RNA with a hairpin structure transcribed only from the repetitive sequence region, while the pre-crRNA is a large RNA molecule transcribed from the entire CRISPR sequence, and the pre-crRNA is cut at the repetitive sequence to form crRNA.
Phase III: Targeted Interference
crRNA and tracrRNA (trans-activated crRNA) in CRISPR/Cas system form chimeric RNA molecules, namely Single guide RNA (sgRNA). SgRNA can mediate Cas9 protein cleavage where it matches the interval sequence, thereby decomposing foreign DNA.
According to different functional components, CRISPR/Cas systems can be divided into Class I systems, Class II systems and Class III systems. These three types of systems can be divided into more subgroups according to their genes encoding Cas protein. Different types of CRISPR/Cas systems have different steps to complete interference.
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