Module 3: Gene Editing by Recombineering With or Without CRISPR-Cas9

Week 3: CRISPR-Cas9-assisted gene editing in

Objectives

To learn the principles and practice techniques on CRISPR/Cas9-assisted recombineering and understand its added effect on E. coli gene editing.

Introduction

Bacterial recombineering relies on the λRed bacteriophage recombination system and short homologies of donor DNA,  specifically single-stranded oligodeoxyribonucleotides (ssODNs), to modify bacterial genomes in a specific manner, such as deletions, knockouts, insertions, and point mutations.1,2,3 It makes conventional cloning with restriction and ligase enzymes seem lengthy and laborious in comparison. However, the recombineering efficiency can still be low in some cases due to a lack of selection markers for selecting edited cells. Screening a large number of cells is often required. Combining CRISPR/Cas9 with recombineering, Jiang et al. were able to significantly enhance the efficiency of gene editing for creating point mutations in E. coli.19

There are primarily two classes of CRISPR systems (Classes 1 and 2), each with different types (types I–VI).31 The type specificity within each class is determined by the specific Cas endonuclease responsible for cleavage and its mechanism of action, and target (DNA or RNA). For example, the effector modules of type I and type III CRISPR-Cas in class 1 are elaborate complexes that consist of multiple Cas protein subunits, while Class 2 systems have a much simpler organization, with the effector module consisting of a single, large, multidomain and multifunctional protein, including the abundant type II (with the effector protein Cas9), and the rare types V and VI, each with a unique architecture of the large effector protein. CRISPR/Cas9 is a Class 2 (type II) CRISPR system that targets phage infection and plasmid transfer and has been utilized as a powerful RNA-guided DNA targeting tool for genome editing. In the wild, CRISPR/Cas9 provides bacteria with sequence-specific adaptive immunity to fight against viruses by integrating short viral sequences into their CRISPR loci, allowing the cell to remember (acquisition/adaptation), recognize (crRNA biogenesis/processing) and clear infections (interference) when encountering the same viruses.32,33,34 Its locus consists of an array of repetitive sequences (repeats) interspaced by short nonrepetitive sequences (protospacers) and a set of CRISPR-associated (cas) genes (Figure 3-5).5,35 The three stages of CRISPR/Cas9 adaptive immunity are the acquisition phase (I), where foreign DNA is inserted into the bacterial genome at the CRISPR locus, and the crRNA biogenesis phase (II), where the inserted foreign DNA is transcribed and processed into crRNA. During interference phase (III), Cas9 endonuclease, associated with the crRNA and tracrRNA forming a protein-RNA complex, cleaves foreign DNA containing a 20-nucleotide crRNA complementary neighboring sequence to the protospacer adjacent motif (PAM) sequence.

Diagram depicting CRISPR-Cas9 process: foreign DNA integration, RNA biogenesis with tracrRNA, crRNA, and Cas9, leading to DNA cleavage.
Figure 3-5. Three stages of a type II bacterial adaptive immunity.35 The locus is an array of repeats interspersed by short stretches of unique protospacers, downstream a set of CRISPR-associated (cas) genes and the trans-activating CRISPR RNA (tracrRNA) gene, which encodes a non-coding RNA with homology to the repeats of crRNA. Upon phage infection, the acquisition machinery (Cas1, Cas2, and Csn2) inserts a new protospacer from the genome of the invading phage into the CRISPR array (Acquisition). The new spacer is transcribed with all other spacers into a precursor CRISPR RNA (pre-crRNA) containing repeats and spacers. The tracrRNA, transcribed separately, anneals to the pre-crRNA repeats before it is processed by RNase III cleavage and further trimming at the 5′ end of the crRNA to shorten the guide sequence to ~20 nucleotides (crRNA Biogenesis). During the final Interference stage, the crRNA–tracrRNA guides the Cas9 endonuclease to cleave foreign dsDNA containing a ~20-nucleotide sequence complementary to the crRNA immediately upstream of a protospacer adjacent motif (PAM) sequence. The PAM and crRNA specify the location where the Cas9 cleaves.

CRISPR/Cas9 cleavage produces double-strand DNA breaks (DSBs), which can result in the loss of large chromosomal regions and lead to serious consequences, including cell death. Two major pathways for their repairs are (i) homologous or homologous-directed recombination (HR or HDR), which requires a homologous template DNA and mostly occurs in the S/G2 phase of the cell cycle, and (ii) non-homologous DNA end joining (NHEJ), in which the DSB ends are directly ligated without a homologous template.

HDR is a repair pathway with accuracy for DSBs facilitated by homologous donor DNA templates, which can be the sister chromatid present during the S/G2 phases of the cell cycle.36 Since the activation of the key protein in the HDR pathway is cell cycle dependent, HDR is restricted in the S/G2 phases of the mitotic cells. HDR also can utilize donor templates to repair directly and thus can be used to make accurate DNA editing with a provided DNA template.

The NHEJ introduces semi-random insertion-deletion mutations (Indels) (Figure 3-6, left).37 However, when a ds- or ssDNA template with homology to the adjacent sequence surrounding the DSBs is available, the HDR pathway proceeds (Figure 3-6, right). The determination of NHEJ versus HDR may be operational despite the conservation of both pathways.38 Their relative contribution to DSB repair varies among eukaryotes. In yeast, HR/HDR plays a dominant role in any DSB repair, whereas NHEJ primarily contributes to DSB repair in vertebrates.39 This is because the molecular mechanism of NHEJ does not require extensive end resection but needs to bind to proteins to protect the ends of DSBs, while HDR requires extensive 5′ to 3′ resection on regions of the duplex to create stretches of ssDNA ends. Therefore, NHEJ only takes ~30 minutes and occurs throughout the cell cycle, while HDR is a precise repair pathway with a complex mechanism that takes more than 7 hours and mainly occurs in the S/G2 phase of cells.

Diagram of CRISPR-Cas9 gene editing. Cas9, guided by sgRNA, makes a double-strand break in DNA. Left, non-homologous end joining causes random mutations. Right, homology-directed repair allows precise gene editing.
Figure 3-6. An overview of non-homologous end joining (NHEJ) and homology-directed repair (HDR). Streptococcus pyogenes Cas9 snips dsDNA 3 bp upstream of the PAM through two distinct nuclease domains of Cas9: an HNH-like nuclease domain cleaving the DNA strand complementary to the guide RNA sequence (target strand) and a RuvC-like nuclease domain cleaving the DNA strand opposite the complementary strand (nontarget strand). After the double-stranded break (DSB), repair proceeds through NHEJ or HDR, depending on the experimental condition. [From Addgene: CRISPR History and Development for Genome Engineering].

The modification frequency of CRISPR/Cas9-mediated NHEJ of mouse genome reaches 60%, whereas HDR is 0.5–20%. HR/HDR and NHEJ are differentially regulated depending on the phase of the cell cycle and the nature of the DSB.38 If a DNA homolog is absent near a DSB during S/G2, then the NHEJ is the only option. In contrast, during the S phase, the sister chromatid is nearby, serving as a homology donor for homologous recombination to perform homology-directed repair (HDR). Unlike eukaryotes, bacteria, including E. coli, are less proficient in NHEJ or lack it completely.38 When NHEJ is absent, alternative end joining (A-EJ) can join the ends using microhomology (usually > 4 bp) and templated insertions of substantial length (> 10 nucleotides). DNA polymerase θ (Pol θ) is the key enzyme for A-EJ.39 Unlike NHEJ, A-EJ is characterized by a large end-resection, mostly due to RecBCD using extensive microhomology and rare DNA synthesis. A-EJ depends on the essential Ligase-A, a distinct end-joining activity that repairs DSBs and generates genome rearrangements, which were reported in pathogenic E. coli and does not rely on key NHEJ proteins absent in E. coli. The acquisition of an antibiotic-resistance gene can occur through A-EJ, and the same goes for the integration of unrelated, nonhomologous exogenous sequences in E. coli.40

After deciphering the mechanism of the CRISPR/Cas9 system, scientists created constructs that express Cas9, tracrRNA, and crRNA to mimic the bacterial sequence-specific adaptive immune system for gene editing. The system has become the most efficient and accurate genome editing tool for various applications across living cells, both prokaryotic and eukaryotic organisms. The three essential components for gene editing are crRNA, tracrRNA, and CRISPR-associated proteins, i.e., Cas9. The crRNA sequence is designed to be paired with the target sequence to be modified. It is complexed with the tracrRNA, which binds to the Cas9 protein to induce DSBs at the target site, followed by the cell’s repair systems to affect deletions, insertions, and other precise gene editing. For example, we can create different CRISPR/Cas9 gene editing systems by changing the target specificity of the RNA-protein complex (tracrRNA, crRNA, and Cas9) without altering the structure of the Cas9 but by altering the sequence of the short crRNA guide. The rapid synthesis of CRISPR for a new target makes the technology versatile for genome editing.35

In addition to gene editing, crRNA-guided Cas9 cleavage can serve as a counter-selection mechanism to eliminate non-edited cells after gene editing, thereby facilitating more efficient screening for edited cells.2,19 Jiang et al. constructed two plasmids to enhance the recombineering efficiency in E. coli (Table 3-5). One is a chloramphenicol-resistant pCas9 plasmid expressing tracrRNA and Cas9, which was placed into E. coli recombineering strain HME63. The other is a kanamycin-resistant plasmid, pCRISPR::rpsL, containing the array of CRISPR spacers with the target sequences to guide Cas9 cleavage of the wild-type unedited cells but not the edited cells with A→C conversion at position #128 of the rpsL allele.19 They also constructed a negative control plasmid, pCRISPR::Φ, without CRISPR spacers and thus no crRNA-guided Cas9 activity for target-specific DNA cleavage (Table 3-5). After placing the pCas9 into the E. coli recombineering strain HME63 expressing three λRed phage proteins,19 they co-transformed the bacteria with ssODN with the A→C alteration at position #128 of rpsL and the pCRISPR::rpsL (Figure 3-7a). Their results show that combining CRISPR/Cas9 with recombineering recovered more edited cells among the treated cells than the control pCRISPR::Φ (recombineering alone). In this lab module, instead of A→C (ssODN) or T→G (antisense ssODN),27 we created a AA→CC (ssODN) or TT→GG (antisense ssODN) two-nucleotide change at the target site to distinguish spontaneous point mutations from recombineering genome editing (Figure 3-7b). The Cas9 cleavage also induces recombination at the target site through HDR. Instead of being killed after binding to the streptomycin to interfere with bacterial protein translation, the properly edited bacteria can grow on the streptomycin media.

Table 3-5 Plasmid constructs in E. coli strain DH5α used in this module19
Plasmid Purpose Antibiotic Resistance
pCas9
(Addgene plasmid #42876)		 Cas9 and tracrRNA expressions chloramphenicol, 25 µg/ml
pCRISPR
(Addgene plasmid #42875)		 No crRNA expression targeting a specific sequence. kanamycin, 50 µg/ml
pCRISPR::rpsL
(Addgene plasmid #44505)		 crRNA expression targeting the rpsL allele. kanamycin, 50 µg/ml

Diagram showing CRISPR-Cas9 gene editing process and DNA sequence comparison. Part a: illustrates plasmids with color-coded genes undergoing electroporation. Part b: highlights DNA sequence changes in rpsL gene, showing mutations with color emphasis.
Figure 3-7. CRISPR-directed editing in E. coli. (a) A pCRISPR plasmid targeting the gene to be edited can be transformed into the HME63 recombineering strain, which contains the Cas9 and tracrRNA expression plasmids. An oligonucleotide specifying a single nucleotide mutation from A to C (indicated by a red circle) is also introduced. (b) A K42T mutation, which confers streptomycin resistance, is introduced into the rpsL. The two nucleotide changes, AA to CC (highlighted in red), and the corresponding amino acid alteration are also indicated in red. [Adapted from “RNA-guided editing of bacterial genomes using CRISPR-Cas systems” by Jiang et al.19]

For this week’s lab, we will compare the efficiency of CRISPR/Cas9-assisted gene editing in E. coli with that of recombineering alone. The λRed-induced E. coli recombineering strain HME63 expressing Cas9 and tracrRNA is electroporated with antisense ssODN and pCRISPR::rpsL (CRISPR/Cas9-assisted recombineering) or pCRISPR::Φ (recombineering alone) (Table 3-5). The editing efficiency is calculated based on the proportion of colonies surviving on streptomycin (recombineering) or streptomycin plus kanamycin (CRISPR/Cas9-assisted recombineering) agar plates. Some of the strepR colonies obtained from both types of colonies will be further analyzed by colony-PCR, agarose gel electrophoresis of the PCR product, and sequence analysis to confirm whether proper gene editing (AA→CC conversion) has occurred.

Instructor Preparation: Creating pCas9-tracrRNA containing HME63 strain

A. Source the recombineering E. coli strains

  1. The instructor must first obtain the recombineering E. coli strain HME63 from the National Cancer Institute’s Center for Cancer Research and follow the steps below to transform these cells with pCas9-tracrRNA plasmid isolated from DH5α, a gift from Luciano Marraffini (Addgene plasmid # 42876; RRID: Addgene 42876)

B. Isolating pCas9-tracrRNA plasmid from DH5α

  1. Grow 5 ml overnight bacterial culture of DH5α containing pCas9-tracrRNA (Addgene #42876) in the chloramphenicol (25 µg/ml) LB medium.
  2. The plasmid DNA is isolated using the Promega PureYield™ Plasmid Miniprep System following the protocols described by the manufacturer.

C. Electrotransformation with pCas9-tracrRNA plasmid

  1. Pick up a single colony of E. coli strain HME 63 to grow in 2 ml LB in a 50 ml conical tube at 30–32°C, 200 rpm overnight.
  2. Add 4 ml LB the following day and continue growing for 3 hr (OD600 = 0.60)
  3. Chill cell at 4°C for 20 min
  4. Pipette 1.2 ml HME63 (600 µl twice) into each of two 1.5 ml microfuge tubes, keeping the tip for the next step.
  5. Centrifuge at 4,000 rpm for 5 mins at 4°C and remove the supernatant by pipetting it to the waste bucket.
  6. Resuspend with 1 ml of ice-cold 10% glycerol, centrifuge at 4,000 rpm for 5 minutes at 4°C, and discard the supernatant by pipetting it into the waste bucket.
  7. Repeat step 6 twice more.
  8. Resuspend the cells in each tube with 60 µl of ice-cold 10% glycerol. Combine the cells in the two tubes into one.
  9. Transfer 50 µl of cells in two 1.5 ml microcentrifuge tubes separately, add sterile RO water with and without 100–150 ng of the isolated pCas9-tracrRNA, and mix.
  10. The mixture is pipetted into a sterile electroporation cuvette as those prepared in Week 1.
  11. Cells are electroporated using a 1 mm Gene Pulser (Bio-Rad) at 1.8 kV, followed by immediately adding 1 ml of room temperature LB medium. (This step is critical.)
  12. Transfer the content to a 15 ml Falcon/culture tube (17 x 100 mm) and shake at 30–32°C (200 rpm) for 2 hr.
  13. Plate 100 µl mixture from each tube onto LB Agar containing 25 µg/ml chloramphenicol at 30–32°C overnight.
  14. Pick a single colony from the ‘with pCas9-tracrRNA’ plate to grow an overnight culture (LB with 25 µg/ml chloramphenicol) to make a 15% glycerol bacterial stock and store it at -70°C.

Procedure

A. CRISPR-assisted recombineering in E. coli

Instructor Preparation on the Day of Lab

The following steps for preparing Cas9-tracrRNA-containing recombineering-competent cells and electroporation are conducted by the instructor:

  1. Streak pCas9-tracrRNA containing recombineering E. coli strain HME63 from a glycerol stock on a 25 mg/L chloramphenicol LB agar plate and grow it overnight at 30–32°C.
  2. Pick an isolated colony and grow it in a 5 ml LB with 25 mg/L chloramphenicol at 30–32°C with shaking (225 rpm) overnight.
  3. Take 0.8 ml of overnight culture to 40 ml of LB (25 mg/L chloramphenicol) in a 250 ml flask (OD600 = ~0.03) at 30–32°C with shaking at 225 rpm until the OD600 is 0.3–0.5 (~3.5–4 hours). [Prepare one flask per two groups.]
  1. For λRed induction, follow those steps described in Week 1.
  2. Add the components listed in Table 3-6 below to one prechilled 1.5 ml microcentrifuge tube according to your group number.
  3. Immediately before electroporation, pipette 60 µl of bacterial suspension into the microcentrifuge tube containing antisense-ssODN and pCRISPR::Φ or pCRISPR::rpsL plasmid, mix by pipetting up and down 3x, and transfer into one prechilled sterile cuvette and electroporate with a MicroPulser Electroporator (Bio-Rad) (1.8 kV and 5 msec (Ecr 1)), followed by immediately adding 1 ml of room-temperature LB medium into the cuvette.
  4. Pipette the electroporated mixture in each cuvette into one 15 ml Falcon/culture tube labeled pCRISPR::Φ or pCRISPR::rpsL and your group number.
  5. Place tubes at 30–32°C with shaking at 225 rpm for ≥ 3 hr. The incubation allows bacteria to recover, perform recombineering (Φ) alone or with the assistance of CRISPR-Cas9 (rpsL), multiply, and express the genes.
  6. Plate bacteria following the instructions in Procedure B.
Table 3-6 Setup for electroporation
Treatment Φ rpsL
Group number 1, 3, 5 2, 4, 6
pCRISRP::Φ (150 ng/µl) 2 µl (300 ng) —
pCRISPR::rpsL (150 ng/µl) — 2 µl (300 ng)
ssODN or antisense ssODN 4 µl (100 pmol) 4 µl (100 pmol)

B. Making Dilutions and Plating

Do not use a vortex, but gently pipette when making dilutions.

  1. Make 102, 104, and 106 dilutions of your electroporated bacteria in LB medium following the steps below.
    1. Take 10 µl from the transformation tube ‘Φ’ or ‘rpsL’ and add 990 µl LB and mix well (102 dilution)
    2. Take 10 µl from (a.) and add 990 µl of LB and mix well (104 dilution)
    3. Take 10 µl from (b.) and add 990 µl of LB and mix well (106 dilution)
  2. Use a spreader to plate 10, 50, and 100 µl of 10-6 dilutions onto three LB agar plates.
  3. Plate 10, 50, and 100 µl of 10-2 dilution from your tube onto three Kan (25 mg/L) LB agar plates.
  4. Plate 10, 50, and 100 µl of the electroporated cells in LB medium onto three Strep (50 mg/L) and three Kan (25 mg/L) + Strep (50 mg/L) LB agar plates, respectively. (Do the LB dilution plates first, use the same spreader for the Kan plate, and get the 2nd spreader for Strep followed by Kan/Strep plates.)
  5. Keep all plates right-side-up as long as possible before flipping them upside-down and incubating at 31–32°C for 24 hr.
  6. Seal plates with parafilm strips, place plates upside-down in the refrigerator, count colonies, and record your data in Table 3-7.
Table 3-7. Bacterial colony (Pay attention to the dilution factor and volume plated)
Treatment pCRISPR::Φ pCRISPR::rpsL
Group number 1 3 5 2 4 6
LB (10 µl of E^6 dilution)
LB (50 µl of E^6 dilution)
LB (100 µl of E^6 dilution)
Kan-25 (10 µl of E^2 dilution)
Kan-25 (50 µl of E^2 dilution)
Kan-25 (100 µl of E^2 dilution)
Strep-50 (10 µl)
Strep-50 (50 µl)
Strep-50 (100 µl)
Strep + Kan (10 µl)
Strep + Kan (50 µl)
Strep +Kan (100 µ)

Data Analysis and Discussion

Show all your calculation steps.

  1. a.    What are the transformation frequencies of kanamycin-resistant bacteria for Φ and rpsL transformation tubes (CFU on the kanR plate divided by the total bacteria plated)?
    1. Is there a significant difference? Why or why not?
  2. a.   What are the editing frequencies of streptomycin-resistant bacteria (CFU on the Streptomycin-containing plate divided by the total bacteria plated) for Φ and rpsL transformation tubes?
    1. Is there any significant difference between the two? Why or why not?
  3. a.   What are the editing frequencies for (i) Φ and (ii) rpsL (CFU on kanamycin plus streptomycin plate divided by total bacteria plated) transformation tubes?
    1. Is there a difference between the two? Why or why not?
  4. a.   What is the fraction of kanR bacteria, which is also strepR (the number of CFU on the kanamycin + streptomycin plate divided by the number of CFU on the kanamycin plate) for (i) Φ and (ii) rpsL transformation tube?
    1. Is there a difference between (i) and (ii)? Explain.

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