CRISPR genome editing has revolutionized genomics research over the past few years, largely facilitated by the CRISPR associated (Cas) enzyme, Cas9. However, occasionally researchers performing genome editing are confronted with a narrow target region that lacks suitable Cas9 protospacer adjacent motifs (PAMs). S. pyogenes Cas9, which is the most frequently used CRISPR enzyme for genome editing, does not efficiently cleave target sequences without its PAM site, NGG [1,2]. Thus, functional target sites may be limited or even non-existent in sequences with low GC content. This poses a significant technical obstacle for editing in AT-rich regions.
Cas12a, a CRISPR endonuclease that recognizes an AT-rich PAM
Cas12a (also known as Cpf1), a putative class II CRISPR endonuclease recently identified and characterized in Prevotella and Francisella , potentially overcomes some of the Cas9 enzyme shortcomings, such as its G-rich PAM requirement. Cas12a endonuclease from IDT is derived from Acidaminococcus sp. BV3L6. It recognizes a T-rich PAM, TTTV, and creates a staggered, double-stranded DNA cut with a 5′ overhang. By including Cas12a in one’s genome editing toolbox, researchers greatly expand the number of target sites available for editing. Not only is this enzyme useful for targeting AT-rich genomes, such as that of Plasmodium falcipracum, but it also has applications in altering disease or phenotype-linked mutations in AT-rich regions through homology-directed repair. In addition, Cas12a does not require a tracrRNA for function.
Cas12a can serve as an alternative to Cas9 endonuclease due to its many unique features. However, Cas12a presents its own challenges. Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, Cas12a exhibits a lower rate of cleavage for the Cas12a PAM sequence. Here we summarize a few findings made by researchers at IDT that provide guidance for improving Cas12a editing efficiency in your own experiments.
Maximize editing efficacy of Cas12a by using TTTV rather than TTTT as the PAM site
Cas12a editing efficacy is PAM-sequence dependent . IDT scientists conducted a comprehensive study that investigated the correlation between choice of Cas12a PAM sites and final editing efficacy. We tested 1240 crRNAs targeting 22 human genes, and found that TTTV PAM sequences (TTTA, TTTC or TTTG) direct DNA cleavage more robustly in comparison to those with TTTT PAM sequences (Figures 1 and 2). It is important to include this principle in your experimental design since, for example, the TTTT motif is significantly more prevalent in the human genome in comparison to its 3 other TTTN counterparts (Figure 3).
Figure 1. Cas12a (Cpf1) genome editing efficacy increases with the use of TTTV PAM sites across 22 human gene exons. The dots represent individual PAM sites ranked by increasing editing activities. TTTN sites from 22 human gene exons were used to design 1240 Alt-R CRISPR-Cas12a crRNAs. HEK-293 cells were transfected with ribonucleoprotein (RNP: Alt-R A.s. Cas12a Nuclease 2NLS complexed with Alt-R CRISPR-Cas12a crRNA). Editing efficiency was determined 48 hr after elecroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required for T7E1 endonuclease assays.
Figure 2. Cas12a (Cpf1) genome editing efficacy increases with the use of TTTV PAM sites in 64 sites of human BCAT gene. The dots represent individual PAM sites ranked by increasing editing activities. 64 TTTN sites in the human BCAT gene were targeted by Alt-R CRISPR-Cas12a crRNAs. HEK-293 cells were transfected with RNP as instructed in the user guide, Alt-R CRISPR-Cpf1—RNP electroporation, Amaxa Nucleofector system. Editing efficiency was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit.
Figure 3. The frequency of TTTN motifs across chromosomes in the human genome. The 4 TTTN motifs and their reverse complements are counted and plotted for each individual human chromosome. The TTTT motif, which is not an effective PAM sequence for A.s. Cas12a (Cpf1), occurs much more frequently than the other 3 motifs (TTTA, TTTC, and TTTG), which are the recommended PAM sequences for A.s. Cas12a.
Enable efficient Cas12a RNP delivery by including Alt-R Cpf1 Electroporation Enhancer
Cell delivery of Cas12a endonuclease appears to be more difficult than for S. pyogenes Cas9. We have yet to identify a transfection reagent that provides robust lipofection of Cas12a RNPs in most of the cell lines we tested. However, electroporation that includes the Alt-R Cpf1 (Cas12a) Electroporation Enhancer serves as an effective alternative for Cas12a RNP delivery to cultured cells. The Alt-R Cpf1 (Cas12a) Electroporation Enhancer is a Cas12a-specific, non-targeting, single-stranded carrier DNA, optimized to work with the Amaxa® Nucleofector® technology (Lonza) and the Neon® Transfection System (Thermo Fisher). It provides increased transfection efficiency, and therefore, increased genome editing efficacy. The Alt-R Cpf1 (Cas12a) Electroporation Enhancer is computationally designed to be void of sequence homology to human, mouse, and rat genomes.
The effectiveness of this reagent has been tested in multiple cell lines derived from major model organisms, including HEK-293, Jurkat, HeLa, and Hep1-6; although, the level of improvement in editing efficiency may vary by cell type. Figure 4 shows targeting of 2 sites in the human and mouse HPRT genes. Their total editing efficiency was compared between groups with or without the addition of the Alt-R Cpf1 (Cas12a) Electroporation Enhancer. The data clearly indicate that Alt-R Cpf1 (Cas12a) Electroporation Enhancer is necessary for efficient Cas12a-mediated editing in RNP electroporation experiments.
Figure 4. Alt-R Cas12a (Cpf1) Electroporation Enhancer is required for efficient CRISPR-Cas12a editing in RNP electroporation experiments. Mouse Hepa1-6 cells and human HEK-293 cells were transfected with 5 µM RNP (Alt-R A.s. Cas12a Nuclease 2NLS complexed with Alt-R CRISPR-Cas12a crRNA) as instructed in the user guide, Alt-R CRISPR-Cpf1—RNP electroporation, Neon system. Electroporation reactions contained either no (dark blue) or 1.8 µM (light blue) Alt-R Cas12a (Cpf1) Electroporation Enhancer. Genomic DNA was isolated 48 hr after electroporation, and total editing efficiency was determined using the Alt-R Genome Editing Detection Kit.
Use of the Alt-R Genome Editing Detection Kit to precisely assess total editing efficiency of CRISPR-Cas12a
As a simple and fast detection method, the T7EI mismatch endonuclease assay has been widely used to estimate genome editing efficiency in CRISPR experiments [4,5]. T7 endonuclease effectively targets and digests mismatched heteroduplex DNA, which results from annealing DNA strands that undergo CRISPR-mediated modifications to a wide-type DNA strand. Assay results are analyzed by visualizing cleavage products and full-length amplicons by gel or capillary electrophoresis.
However, T7 endonuclease is less robust in recognizing single-base mismatches . Previously, we have shown that the T7EI assay underestimates the efficiency of genome editing mediated by CRISPR-Cas9, when compared to next-generation sequencing (NGS) approach, which captures all editing events regardless of type . Interestingly, in Cas12a edited samples, the editing efficiencies estimated by T7EI are much closer to the corresponding NGS results, suggesting that T7EI is more accurate in quantifying on-target genome editing events for CRISPR-Cas12a (Cpf1) (Figures 5 and 6).
IDT makes the reagents and protocol for ourT7EI mismatch endonuclease assay available as the Alt-R Genome Editing Detection Kit. Use it to detect on-target genome editing and estimate genome editing efficiency in CRISPR-treated cells.
Figure 5. Genome editing efficiency estimated by T7EI assay parallel NGS results in 8 Cas12a (Cpf1) edited PAM sites in the human HPRT gene. Alt-R CRISPR-Cas12a crRNAs (30 nM) were introduced by lipofection into HEK-293 cells that constitutively express A.s. Cas12a nuclease. 8 PAM sites in the human HPRT gene were targeted. To assess editing efficiency, genomic DNA samples were harvested 48 hr after transfection and tested using the Alt-R Genome Editing Detection Kit (dark blue bars). The same DNA samples were also analyzed using targeted NGS (light blue bars). Amplicons were run on a MiSeq® system (Illumina) and sequencing data were analyzed using a proprietary data processing program developed in-house. Error bars represent standard deviation for triplicate lipofection experiments. The correlation coefficient for the two analysis is shown as the R value.
Figure 6. Genome editing efficiency estimated by T7EI assay parallel NGS results in 3 Cas12a (Cpf1) edited PAM sites across each of 4 human genes. Alt-R CRISPR-Cas9 crRNA (30 nM) were introduced by lipofection into HEK-293 cells that stably express S. pyogenes Cas9 nuclease. 3 PAM sites were targeted in each of the 4 genes, BCAT, ALDH2, BIRC5, and AR. Similar to the experiment described in Figure 4A, genomic DNA was isolated 48 hr after lipofection, and was tested using both the Alt-R Genome Editing Detection Kit and amplicon sequencing.
What we have learned about CRISPR-Cas12a genome editing
- With the ability to recognize T-rich PAM sites, Cas12a further expands the number of available target sites for genome editing. This feature can be especially useful when design space is limited.
- When designing CRISPR-Cas12a experiments, we recommend testing 3 or more crRNAs per target gene. For optimal results, we suggest designing Cas12a guide RNAs that use TTTV (i.e., TTTA, TTTC, or TTTG) instead of TTTN as the PAM site.
- Compared to Cas9, which efficiently cleaves DNA when targeted using most potential NGG PAM sites, Cas12a is less potent in introducing double strand breaks when DNA is targeted using the TTTV PAM sites.
- The Alt-R Cpf1 (Cas12a) Electroporation Enhancer is critical for optimal delivery of Cas12a RNPs by electroporation and is recommended for experiments using this delivery method.