The RNA-guided nature of CRISPR nucleases (e.g., Cas9, Cpf1) makes them highly amenable to multiplex applications, including multi-gene knockout and large-scale genomic modifications. This article — part 1 of a two-part series on multiplex CRISPR gene editing — discusses these applications and outlines practical considerations for multiplex CRISPR gene editing experiments.
Why Multiplex CRISPR?
There are many CRISPR applications that benefit from Multiplexing, including multi-gene knockout, large-scale genomic modification, and CRISPR genome editing with reduced off-target activity.
Multi-Gene Knockout (KO)
Multi-gene KO is the most obvious and conceptually straightforward application of multiplex CRISPR gene editing. Simply deliver more than one guide RNA (each targeting a different gene) to your cells along with a CRISPR nuclease. This process, however, is not so straightforward in practice because the editing efficiency of a double KO will be invariably lower than a single KO. Lower efficiency means you will need to screen more clones to obtain one with a double KO. Triple KOs and higher are certainly possible with CRISPR. However, they may require multiple rounds of gene editing, or significant optimization, to accomplish.
Multi-gene KO is particularly useful for targeting redundant genes, or when weak phenotypes are observed with single gene KO. 
Large-Scale Genomic Deletions
CRISPR nucleases can be used to make large-scale genomic deletions, including gene excision and chromosomal deletion.  Editing on this scale requires at least two DSBs that flank the targeted genomic region. For such applications, you can use either multiple gRNAs that target unique genomic sites, or a single gRNA that targets a repeated sequence in the targeted genomic region (i.e., a single gRNA that targets multiple sites). The former approach is better suited for more precise deletions, such as exon or full gene excision. For chromosome-scale deletions, the latter approach often provides better efficiency. 
When using multiple gRNAs that target unique genomic sites, you can improve your editing efficiency by using a dual-paired gRNA approach (i.e., two pairs of gRNAs that flank the targeted genomic region). [3, 4] However, you might experience diminishing returns when using more than four gRNAs, as the efficiency of individual gRNAs can decrease as you increase the number of gRNAs delivered simultaneously. Given the relatively relaxed PAM constraints of commonly used CRISPR nucleases (e.g., Cas9), finding multiple such paired gRNAs is generally possible, although your mileage may vary depending on which nuclease you use and your targeted genome.
In general, the distance between the binding sites of the different gRNAs influences gene editing efficiency for large-scale deletions. The greater this distance is, the lower your efficiency will be.  This likely depends on the timing of each DSB. If one DSB is repaired before the other is initiated, then the intermediate fragment will not be excised. 
Reducing Off-Target Cas9 Activity
Multiplexing can also be used to reduce off-target effects in single KO applications.  Typically, this involves using a variant of Cas9 that can produce a DSB only when two are present in close proximity. There are two primary tools available to accomplish this:
- Cas9 nickase : An engineered variant of Cas9 with partially ablated nuclease activity (alanine substitutions at either D10 or H840/N863 residues)
- dCas9-FokI dimers : An engineered variant of Cas9 with fully ablated nuclease activity that has been fused to the endonuclease domain of the restriction enzyme FokI.
Both of these tools work only when paired and require two unique gRNAs to initiate a DSB. An individual Cas9 nickase would only nick a single strand of DNA, and an individual dCas9-FokI would not exhibit any nuclease activity at all, partial or not. This is because the nuclease activity of FokI requires that it form a homodimer with another FokI endonuclease domain. To be precise, paired dCas9-FokI enzymes must bind genomic sites no further than 15-25 base pairs from one another to work . For Cas9 nickase, gRNA binding sites should be <20 bp apart under ideal circumstances, but can work when as far as 100 bp apart .
Both of these tools drastically reduce off-target effects because it is extremely unlikely that any two off-target sites will be in close enough proximity to one another to generate off-target DSBs. However, there is a trade-off, as this strategy also yields lower on-target efficiencies than normal Cas9.
While some applications — such as multi-gene KO — absolutely require multiplexing, others do not, at least not always. For example, although Cas9 nickase can reduce off-target effects, this is not always necessary. After all, some gRNAs are very specific! And some applications don’t necessarily require high specificity. But for those that do — e.g., therapeutic applications or generating KO mice — the need for higher specificity outweighs the cost of lower efficiency.
Of course, some technological advances have been made to boost the advantages of multiplexing while mitigating its disadvantages. These advances are discussed in Part 2 of this two-part series: CRISPR Multiplexing Strategies: What’s The Choice?
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