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).
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.
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.
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.