Metagenome extraction method for removing host genomic DNA based on CRISPR-Cas

Abstract

The invention discloses a metagenome extraction method for specifically reducing or removing a host genomic DNA based on a CRISPR-Cas system composition. The invention aims to provide a technique forreducing a host genomic DNA background in metagenome sequencing and improving effective sequencing data of other microorganisms. The CRSIPR-Cas system in the method comprises a plurality of crRNA or crRNA/tracrRNA derivatives, as well as a biotin-labeled Cas protein or variant protein with endonuclease activity deficiency. The sequence of the crRNA or crRNA/tracrRNA derivatives is complemented with a repetitive sequence Alu element region (target sequence) in the host genomic DNA, so that the binding of the Cas protein or the variant thereof with endonuclease activity deficiency is guided to be bonded on the host genomic DNA, the CRISPR-Cas system and the bonded host DNA are captured by adding magnetic beads coated with streptavidin, and thus the concentration of the host genomic DNA in asample solution is reduced and the abundance of nucleic acids of other non-host genomic DNA is improved.

Description

Technical field [0001] The invention belongs to the field of metagenomic detection, and specifically relates to CRISPR technology and a method for subtracting host genomic DNA in metagenomic nucleic acid samples. Background technique [0002] The clusters of regularly spaced short palindrome repeats (CRISPR) / CRISPR-associated protein (Cas) system is an adaptive immune defense mechanism generated by bacteria and archaea in the process of continuous evolution to protect their own genomes from Interference and destruction of foreign nucleic acids such as bacteriophages and viruses. The CRISPR / Cas system is an adaptive immune system. It uses CRISPR RNA (crRNA) to guide the Cas protein to recognize the invading foreign genome in the form of base complementation and to shear its DNA. According to the sequence and structure of Cas protein, the CRISPR / Cas system is divided into type I, type II and type III. Both type I and type III systems require multiple Cas protein elements, while t...

AI Technical Summary

Problems solved by technology

Existing methods for removing host nucleic acids include (1) removal based on the use of DNA / RNA probes targeting host DNA, usually requiring denaturation of the sample nucleic acid followed by hybridization incubation, the hybridization process takes a long time and is costly; (2) Based on the principle that methylated CpG binding domain proteins (such as MBD1, MBD2, MBD3, MBD4 proteins) can be combined with methylated CpG islands widely present in eukaryotic DNA, the removal efficiency is usually low and the effect is not ideal , and will also lose some of the nucleic acids of other non-host eukaryotic organisms such as fungi or parasites; (3) based on the size difference between the host cell and the microorganism or other physical differences (such as sedimentation velocity), the removal efficiency of the host cell is usually very high Low, free host DNA cannot be removed, and nucleic acids of fungi, parasites, and some intracellular microorganisms will also be lost; (4) Based on the fact that human cells are more fragile than microbial shells, use hypotonic solutions, or mild detergents or Other enzymatic treatments that only destroy host cells and then use membrane-impermeable propidium iodide azide (PMA) to modify host DNA exposed to solution

PMA is a photoreactive dye with high affinity for DNA. When exposed to strong visible light, the dye intercalates into double-stranded DNA to form a covalent link, forming chemically modified DNA. At this time, it can no longer be amplified and complete subsequent sequencing library construction. process

This method has high removal efficiency but also has the disadvantages of complex steps and the removal of dead bacteria DNA at the same time

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