Strategies for gene editing in bacteria combining CRISPR/cas9 and λ-Red systems

CRISPR/cas9, a gene-editing tool, has been widely used in site-directed mutagenesis in eukaryotic cells and especially in mammalian system. Contrastly, in bateria genes editing, CRISPR/cas9 has been rarely developed. For one thing, double strand DNA break triggered by Cas9 is lethal to bacterias which is lack of the solid double strand DNA damage repair system. For another, bacteria has a low intrinsic frequency of recombination. To address these hurdles, the following protocol will combine traditional λ-Red recombinase and CRISPR/cas9 to apply in bacteria genes editing. As is illustrated in Figure 1, CRISPR/cas9 in bacteria is responsible for site-specific cutting and negative slection for those bacteria without recombination. Adding λ-Red recombinase to the system can acclerate bacterial gene editing and survive. This protocol can apply to genes knock in and knock out with approximately one hundred percent efficiency. Especially competent and convinent for gene knock out, it can easily knock out three genes at the same time.

Fig 1. CRISPR/cas9 and λ-Red mediated genes editing in bacteria

CRISPR/cas9 and λ-Red mediated bacterial genes editing tools contains two plamids: pCas and pTarget. pCas is derived from pKD46, which is introduced Cas9 into the pKD46. Thus, like pKD46 with temperature sensetive replication origin, the optional temperature for proliferation of pCas plasmid in E.coli is 30℃, not 37℃. pTarget is responsible for carrying the guide RNA and DNA donor template into bacteria, which is dirven by lacIq-Ptrc promoter. Sure, pCas and pTarget have distinctive antibiotic resistance for selective of the bacteria with two plasmids. As is illustrated of Fig.2 and Fig.3, pCas has kanamycin resistance, while pTarget has Spectinomycin resistance.

Fig 2. Map for pCas

Fig 3.  Map for pTarget

Forward primer:  - (N) ₂₀ GTTTTAGAGCTAGAAATAGC

Reverse primer:  - (N) _20R ACTAGTATTATACCTAGGAC

2.2 insert the guide RNA into pTarget by inverse PCR
High fidelity and time-saving DNA polymerase is used for inverse PCR. Fast pfu is the first choice. In 50 μl PCR system, add 10-20 ng pTarget as PCR template. The length of PCR product is about 2200 bp. All the PCR reaction system and condition are followed as the manual of the distinctive high fidelity DNA polymerase producers.

Note: the cycle of PCR is usually from 22 to 28

2.3 Transformation

Recover the PCR product using the PCR clearance kit. Then, to eradicate the pTarget template, add two 2 units of DpnI (NEB) to digest at 37 ℃ for 1 hour. Next, transform the above PCR product into E. Coli (DH5α or Top10) and culture overnight on the LB plate at at 37 ℃.

2.4 postive clones identification
Pick single clones from LB template and culture for several hours in shakers at at 37 ℃, perform PCR to identify the positive clones. Use the above reverse primer (N20R---) and Target-F as primers, and the PCR product is about 500 bp. Selected positve clones are confirmed by sequencing. MoClo-F is primer for sequencing.
Target-F: CGACCTACACCGAACTGAGA
MoClo-F: AGCGAGGAAGCGGAAGAGCG

3. Manipulate gene knockout in bacteria

Transform pCas into the interest bacteria, culture on relative culture plate with the kanamycin (30-50 mg/L) at 30 ℃. Pick up single clones into liquid LB, shake at 220 rpm, 30 ℃. Induce λ-Red expression with 10mmol/L arabinose when OD600 Value of the above culture reach about 0.2, then OD600 Value move up at 0.4-0.5, collect the bacteria by centrifugation and prepare competent cells for electrotransformation. Next add 80ng pTarget F (knock out) and 400 ng DNA Fragment with homologous arms into 50 μl of the above competent cells. Mix evenly, transfer into the precold 1mm electrotransform cup and do the transformation at 1.8 KV using Bio-Rad electrotransformation equipment. Last, revive the above cells at 30 °C, 180 rpm by shaking for 1 hours, selectively culture on plate containing 30-50 mg/L kanamycin and 50mg/L spectinomycin, overnight at 30 °C. The following day, Pick the single clones, culture at 30 °C for about 12 hours, induce cas9 expresson by IPTG (0.2 -0.5mM) for about 12 hours. Next, culture at 37 °C for curing pCas plasmid.

4. Interest bacteria identification

To determine whether the genome of bacteria has been edited(knock out and knock in ), please do PCR identification.

4.1 Extract the bacteria genomic DNA as template or boiled the cultured bacteria as template directly.

4.2 Perform the PCR assay using WT bacteria as constrol, assess the knockout by comparing PCR length with WT counterpart or T7E1 assay.

Fig 5. Flowchart for gene editing in bacteria using the pCas and pTarget

5. Generate pTarget carrying multi guide RNA for distinctive genes editing at same times

Strategies for construction of pTarget with muti-gRNA by using the isocaudomer restriction enzyme character of BamHI and Bgl∥. As illutrated as following, BamHI and Bgl∥ can produce the homo-cohensive end with the same sequence (GATC).

BamHI:

BglII

Suggest to knock out/ in at most of three genes.

1. Generation of three ptarget-gRNA by inverse RNA following the above method for the first step.

2. Digest the pTarget-gRNA2 and pTarget-gRNA3 with BamHI and Bgl∥ , recover the fragment with guide RNA by gel purification kit. Similarly, digest pTarget-gRNA1 with BgIII, ligate the gRNA2 into pTarget1 to get pTarget-gRNA1-gRNA2. Then, digest the pTarget-gRNA1-gRNA2 with BgIII, ligate gRNA3 into pTarget-gRNA1-gRNA2 to make pTarget-gRNA1-gRNA2-gRNA3.