CRISPR-CAS 9

CRISPR-Cas 9 systems contain two components: a guide RNA and a Cas 9 protein. The gRNA is a short synthetic RNA that defines the genomic target to be modified and the Cas9 protein is a DNA endonuclease. These two components will form a complex which recognizes the PAM site and binds to the target site, and then uses the cas9 protein to cut the double-stranded DNA in the target region to break it, inducing cells to perform homologous recombination repair to achieve the purpose of gene knockout or knockin.
It is one of the most scalable genome editing technologies due to the ease of generating gRNAs. Simply changing the target sequence present in the gRNA can alter the genomic target of the Cas protein thus enabling the knockout of different genes.

Our knockout method

Figure1: Four-step method to knock out E.coli AIS strain

We refer to the article published by Qi Li, Bingbing Sun and other people in 2020.
Step 1: Preparation of competent cells and pEcCas transformation
Transfer pEcCas into competent cells of the target strain through chemical transformation
Step 2: Construct pTartget plasmid and obtain donor DNA
pTarget plasmid: Ncbi obtains the gene sequence to be knocked out, and chopchop is used as a tool to find gRNA, which is generally 20 bp. The 20 bp gRNA was assembled by Gibson to construct the pTarget plasmid. Donor DNA: Design and produce donor DNA containing homologous arms. Let E. coli perform homologous recombination repair, construct upDNA and downDNA through one round of PCR, and assemble upDNA and downDNA through two rounds of PCR to construct donor DNA.

Figure2: The design of ptarget plasmid(A) and donor DNA (B)

Step 3: Place ptarget and donor simultaneously into E. coli in step 1 through electroporation to achieve knockout of the target gene. (See protocol for methods)
Step 4: Curing plasmid
Positive clones with successful knockout were identified by colony PCR.
a. To cure the pTarget plasmid, we put the positive clone into LB liquid medium containing rhamnose and kanamycin, culture it for 6 hours, and then transfer it to the medium without antibody at a ratio of 1:100;
b. Curing for pEcCas plasmid, we add sucrose to the liquid culture medium, culture it overnight, and streak it onto the LB plate containing sucrose without antibody the next day;
c. Screen the colonies on LB plates with kanamycin, LB plates with spectinomycin and LB plates, colonies that grew only on LB plates were cured both pEcCas and pTarget.

Our achievement

Different from the traditional DH5-a and BL21 strain, we successfully used CRISPR-Cas9 technology in the Ais strain (E.coli CICC20905) and achieved the knockout of three genes and the successful elimination of the plasmid. Ais strain has not been used in previous igem competitions, and few people have studied it. Therefore, we provide a complete set of guidelines for the use of gene knockouts for future igemers.

Figure3: Knock out genes in AIS strains. （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout. (B)colony PCR to respectively determine the knock-out of rhtA、ilvIH、ilvBN.（C）verified the knock-out result through the sequencing testing. (D)three plates carrying different antibiotics to confirm the cure of pTarget and pEcCas plasmid

Contribution part

Code number	Name	Description	Characterization
BBa_K4808003	g-rhtA	target the rhtA gene	✅
BBa_K4808004	g-ilvI	target the ilvI gene	✅
BBa_K4808005	g-ilvB	target the ilvB gene	✅
BBa_K4808009	pTarget	Carry specific guide RNA	✅
BBa_K4808011	pEcCas	Carry cas9 protein	✅

reference：
Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036