As Kendall mentioned in Tuesday's blog post, keeping up with the newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions to make, it makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots of questions.
To keep up with all these questions, Dr. Le Cong and Dr. Feng Zhang developed a Google Forum to allow scientists to ask questions and get answers on the fly. But just like the growing PubMed article list, the Genome Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate these FAQs, Le Cong worked with Addgene Senior Scientist, Matt Ferenc, to organize the CRISPR forum's most popular questions and answers.
Here are just 4 of the CRISPR-Cas9 FAQs. If you still have more questions after reading through these, we've got 12 more Q&As which focus on genome editing using the Zhang lab's CRISPR/Cas plasmids.
Designing Your CRISPR Genome Editing Experiment
Q1: Should I use wildtype or double nickase for my CRISPR genome engineering experiments?
A1: When assessing which nickase type to use for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency but has been shown to have off-target effects. 'Double nickase' is a new system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design but with better accuracy (in other words, lower off-target effect.
The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, et. al, 2013 Science paper. For example, if you want to use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase).
The concept of the double nickase system is that you can express two different chimeric gRNAs with the Cas9 nickase which will together introduce cleavage of the target site with efficiency similar to using a single chimeric gRNA. At the same time, the off-target effects are reduced because the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does. There are a few references for the double nickase system, including one recently from the Zhang group.
Learn more here.
Q2: When designing oligos for cloning my target sequence into a backbone that uses the human U6 promoterto drive expression, is it necessary to add a G nucleotide to the start of my target sequence?
A2: The human U6 promoter prefers a 'G' at the transcription start site to have high expression, so adding this G couldhelp with expression, though it is possible for the plasmid to still express without the G. Because the G is only one base, the Zhang lab usually adds it when they order the oligo. If your spacer sequence starts with a 'G', you naturally have one and do not need to add an additional 'G'.
Q3: What is the maximum amount of DNA that can be inserted into the genome using CRISPR/Cas forHomologous Recombination (HR)? How long should the homology arms be for efficient recombination?
A3: The most we've tried to insert so far has been 1kb. We used homology arms that were 800bp long.
Tips for Using CRISPR-Cas9 at the Lab Bench
Q4: After the introduction of a mutation into the genome, how can cells with that mutation be selected/screened?
A4: Before starting your experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected. Alternatively, you can use a selection marker to select transfected cells (for example, plasmid with a puromycin resistance cassette, such as PX459). After you co-transfect the CRISPR/Cas system with your homologous recombination (HR) template, you could then:
- Confirm your HR by doing Restriction Fragment Length Polymorphism (RFLP) (see Figure 4 of the Cong, et. al, 2013 Science paper).
- If you detect positive HR, isolate single-cell colonies, grow them up, then perform individual genotyping (using Sanger sequencing, for example) on each colony in order to screen for positive ones.
-- OR --
- If your HR template has a selection marker such as puromycin, you can (also) select for the positive colonies by puromycin selection. You could then confirm this purification by performing a genotyping assay (such as Sanger sequencing).
More FAQS, CRISPR Protocols, and gRNA Design Tools
Need more questions answered about CRISPRs?
- Check out the full list of 16 FAQs answered by Le Cong
- Read Addgene's CRISPR guide for background information on CRISPR/Cas9 systems
- Peruse the most recent genome editing review articles, such as: Sander JD & Joung JK, Nature Biotech, 2 March 2014.
- Or browse the articles related to the most frequently requested CRISPR plasmids at Addgene
Find protocols and gRNA design tools:
- List of CRISPR protocols developed by a variety of labs and optimized for specific plasmids
- Links to different software to help you identify your gRNA target sequences
Additionally, I would like to thank Le Cong for all his hard work creating amazing resources for the scientific community and sharing these resources with Addgene. Thank you, Le!