The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.
Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism’s genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.
To study the basis of normal physiology and disease pathology, there is a need to precisely manipulate gene expression in model organisms. For mammalian model organisms, this is largely centered on the creation and development of transgenic mice wherein a genetic element of interest is flanked by sites recognized by a recombinase. This can result in a site specific manipulation of these flanked genes. While this has been a successful strategy, it is time and resource intensive; for example, creating a triple transgenic animal that would express a floxed gene, Cre recombinase, and a Cre reporter gene requires multiple matings and validation. In contrast, the stereotaxic injection of replication defective viral particles encoding a fluorescent protein and the recombinase into a floxed gene animal does not require complex genotyping or breeding strategies1. Further, if a fluorescent protein and Cre expressing virus is co-injected with a second virus encoding a different fluorescent protein, then this provides a within-tissue control for the targeted genetic manipulation. While this strategy still requires the use of knock-in animals, virally mediated RNA based strategies circumvent the need for transgenic animals. For example, stereotaxic injection of replication-deficient viruses that encode a fluorescent protein and a short hairpin RNA (shRNA) can use the cell's endogenous RNAi machinery to result in a potent reduction of the transcript of a gene of interest. However, shRNA strategies produce subtle gene knock-downs often resulting in modest cellular phenotypes2. While a knock-down may be more physiologically relevant for heterozygous gene dysfunction, its decreased robustness compared to a knock-out is not ideal for phenotypic discovery of novel genes.
A third technique that has recently emerged, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9 (CRISPR-associated protein 9) system, relies on the expression of both a small exogenous RNA and a DNA cutting enzyme. The CRISPR/Cas9 system was adapted from the prokaryotic immune system which evolved a method of identifying foreign, invading DNA from viruses and targeting it for degradation via the Cas9 enzyme3,4. This powerful genome editing technique can be used for creating targeted deletions, insertions, and mutations; and the following protocol will outline how to make deletions in a gene of interest in order to knockout its expression in vivo. The Cas9 enzyme must be expressed with a guide RNA homologous to the region of interest and contiguous with a scaffold RNA. Knockout of a gene using this technique requires targeting Cas9 to a specific region in the genome using synthetic guide RNAs (sgRNA), and inducing double stranded breaks (DSBs) at a site of interest. These DSBs are then repaired by the endogenous cell-repair machinery via non-homologous end-joining (NHEJ) which lead to indels that may produce missense or nonsense mutations and can therefore create a loss of functional protein expression5. Because this system produces genomic alterations, it only requires the transient expression of the Cas9 and sgRNA. However, it is desirable for a stable fluorescent indicator to identify cells and their progeny manipulated in this manner.
Lenti- and retroviruses have the advantage of stably integrating DNA of interest into host cells which maintain long-term expression and are passed down to daughter cells during mitosis. This protocol describes the design and production of two types of replication defective, high titer retroviruses: the human immunodeficiency virus derived lentiviral particles (lentivirus) and those based on murine Maloney Leukemia virus (retrovirus). While both of these viruses are capable of supporting stable expressing of large transgenes, the retroviral particles can only integrate into the genome during cell division with the degradation of the nuclear envelope, and therefore can be used as a tool to label and birth-date cells6. While lentiviruses have a reputation for being relatively low titer7, this methodology, including the use of caffeine8 during viral collection, routinely produces titers of 109 and 1010 particles/ml. Another advantage of lenti- and retroviruses is the tolerance for very large inserts. The following collection of protocols outlines the procedure for designing a lenti or retrovirus encoding a fluorescent reporter, sgRNAs, and Cas9 to utilize the CRISPR/Cas9 system to modify DNA as well as express a fluorescent protein.
Mouse stereotaxic neurosurgery is a valuable method for injecting viruses in vivo to study morphology, function, and connectivity of infected neurons. Viral infection in neurons can be used to manipulate expression levels over an extended period of time, such as throughout development, and expression can be precisely controlled by the use of various drug inducible systems and specific Cre driven expression. This particular protocol explains how to inject a virus expressing an sgRNA and Cas9 to knockout a gene of interest in the brain of an adult mouse. Mice recover very quickly from this procedure and expression of the viral transgene can be seen within 48 hours post injection. However, fluorophore expression appears to increase over the course of weeks resulting in near maximal levels by 3 weeks post-infection. Mice that undergo viral stereotaxic injection can be used for behavior, electrophysiology, or morphological studies. Overall, the purpose of these procedures is to demonstrate how to knockout a gene in the adult mouse brain using stereotaxic surgery and a virus expressing a specific sgRNA and Cas9.
There are a few critical steps that are important for successful viral packaging. Cell health is critical before and during the transfection, as unhealthy cells will greatly reduce the amount of virus produced. If the transfection and packaging are successful, then 100% of the cells should express the fluorophore and the cells should form a functional syncytium. In step 3.2.4, tapping the tube is necessary for high-titer transfection efficiently, and the pH of the HEPES-buffered saline must be exact. The maxi-preps that produce the plasmids necessary for viral packaging must be extremely pure. To this point, it is helpful to ethanol precipitate the final DNA elution and re-suspend in Tris-EDTA buffer. It is also very important to reduce the amount of serum to 2% or less in the media that the caffeine is added to on Day 6 (step 3.4) before viral collection. If the serum is not reduced, then the final purified virus will contain an undesirable amount of serum protein. The use of polyethylene glycol 6000 when precipitating the viral particles excludes the necessity of ultracentrifugation. It is also important to note that the Cas9 CRISPR containing viruses typically have a titer around 10 fold less than viruses solely containing a fluorophore.
For the stereotaxic surgery, the use of inhaled anesthesia allows rapid and precise control or the animal's consciousness compared to injectable anesthetics and allows anesthesia over a larger age range. It is highly important to keep the surgical instruments clean and sterile, and reproducible targeting requires precise positioning of the head. Make sure there is no pitching or rolling of the head in the stereotaxic instrument and that the skull feels firmly in place. It can be useful to allow the skull to dry in order to find the sutures to determine the stereotaxic coordinates. Also, the rate and volume for each stereotaxic coordinate should be empirically determined.
This technique is limiting in that the spread of a lenti- or retrovirus is restricted, especially when compared to adeno-associated viruses (AAVs).Therefore, these viruses are valuable when infecting a discrete brain region, but not for overall infection associated with AAVs used for behavioral analysis in animals. The use of caffeine in this protocol greatly increases the titer of these viruses, but they are still not as high as the titers achieved in AAV packaging. Also, stable integration is only an advantage of fluorophore expression, as CRISPR/Cas9 forms stable genomic edits even when transiently transfected and it is possible that on-going expression of the Cas9 and sgRNA may eventually produce off target effects. The transient expression the CRISPR/Cas9 system with AAVs is sufficient to produce genomic changes that are propagated throughout cell divisions, however, fluorophore expression will not be maintained.
Creation of lenti- and retroviruses utilizing the CRISPR/Cas9 system will impart the ability to target any novel gene in a wide variety of organisms. The efficiency of gene editing appears to be dependent on the sequence of the guide RNA targeting the Cas9 cleavage. It has been empirically determined that between 10% and 80% of clones contain indels after sequencing infected Neuro2A cells. It is presently unknown whether indel frequencies calculated in Neuro2A cells are reflective of those in neurons. Guide RNA design software such as Benchling now include an "on-target" score that may be able to predict the efficiency of a given target sequence. To what degree such "on-target" scores are reliable needs to be empirically determined in neurons and other cell-types as the CRISPR-Cas9 system becomes more widely implemented.
Lentivirus-based transgenic animal production has been variably successful with reports that the lentivirus-delivered transgenes become silenced11. CRISPR mediated gene editing of DNA may be passed through the germ line to generate whole animal models. Thus, stable genomic editing may be achievable despite the silencing of viral-delivered fluorophores and Cas9 transgenes. This may provide an efficient platform for targeted genomic alterations. The viral delivery of the CRISPR/Cas9 system, while not requiring transgenic organisms, is complementary to those techniques. For example, injecting such viral particles into a compound transgenic animal that inducibly expresses Cre and Cre dependent opto- or chemo-genetic transgenes should facilitate complex studies into the relationship between genetic manipulations and neuronal activity. A second example is to deliver these Cas9/sgRNA viral particles into a conditional knockout in an attempt to screen for gene-gene interactions. Finally, another exciting route of this research is the screening of phenotypes and therapeutic compounds in patient derived cells, which can be used to validate and discover genetic networks that are disrupted in various diseases.
The authors have nothing to disclose.
This work was supported by the NINH grant R01MH097949 and the Autism Speaks Pilot Grant 7359 to BWL and the Norris Cotton Cancer Center Optical Imaging Shared Instrumentation Grant P30CA023108.
List of Cell Culture Reagents | |||
293FT cell line | Life Technologies | R700-07 | For Lentivirus |
293GP cell line | Clontech | 631458 | For Retrovirus |
Iscove's Modification of DMEM (IMDM) | Corning | 10-016-CV | Complete IMDM with 10% FBS, 1% NEAA, 1% L-Gln, and 1% P/S |
Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
MEM Nonessential Amino Acids (NEAA) | Corning | 25-025-CI | |
L-Glutamine solution, 100X (L-Gln) | Corning | 25-005-CI | |
Pennicillin/Streptomycin solution, 100X (P/S) | Corning | 30-002-CI | |
Polystyrene 10cm plate | USA Scientific | CC7682-3394 | |
Trypsin EDTA 1X | Corning | 25-053-CI |
|
Reagent | Company | Catalog Number | Notes |
List of Transfection Reagents | |||
5ml polystyrene tubes | Fisher Scientific | 352054 | |
Calcium Chloride Dihydrate (CaCl2) | Fisher Scientific | C69-500 | Make a 2.5 M solution in ddH2O |
Sodium Chloride (NaCl) | Fisher Scientific | S271-3 | |
HEPES | Fisher Scientific | BP2939-100 | |
Sodium phosphate dibasic | Fisher Scientific | S369-500 | |
(Na2HPO4) | |||
2X HEPES Buffered Saline (HBS) | 500ml: 8.2g NaCl, 5.95g HEPES, 0.106g Na2HPO4, pH 7.01 (exact!) | ||
filter and store at 4°C | |||
Caffeine | Sigma-Aldrich | C0750-5G | |
0.22 µM syringe filter unit | EMD Millipore | SLGV033RS | |
0.45 µM syringe filter unit | EMD Millipore | SLHP033RS | |
60CC L/L Syringe | Med-Vet International | MV60CCLL | |
50 ml Conical Tube | Corning | 352098 | |
polyethylene glycol 6000 (PEG 6000) | Millipore | 528877 | |
(10X) Phosphate Buffered Saline (PBS) | National Diagnostics | CL-253 | |
0.5 ml microcentrifuge tubes | USA Scientific | 1605-0000 | |
Matrigel | Fisher Scientific | CB-40230A | |
6-well plate | Fisher Scientific | 353046 | |
Paraformaldehyde | Fisher Scientific | AC41678-5000 | |
Donor Horse Serum | Cellgro | 35-030-CV | |
TritonX-100 | Sigma-Aldrich | X100-500ML | |
10ml serological pipette | Fisher Scientific | 357551 | |
anti-GFP, rabbit, 488 conjugate | Invitrogen | A21311 | |
Reagent | Company | Catalog Number | Notes |
Stereotaxic Surgery Reagents | |||
Vet-Syringe vet use T.B. Syringe only 1cc luer slip T.B. 100/bx | Med-Vet International | 1CCVLS | |
Isoflurane | |||
Stainless Steel Scalpel Blades, #10, 100-pk | Med-Vet International | JOR580S | |
artificial tear ointment | Med-Vet International | RXPARALUBE-O | |
PVP PrepSolution | Med-Vet International | HPIV108208H | |
normal saline | Med-Vet International | DYND500MLSH | |
cotton tipped applicators | Med-Vet International | CTA6 | |
Triple antibiotic ointment | Med-Vet International | RXTRIP-OI15 | |
MONOJECT® Needles Soft Pack 25g x 5/8" | Med-Vet International | 25058 | |
6-0 silk sutures | Med-Vet International | MV-711 |