Alt-R™ CRISPR-Cas editing systems
CRISPR genome editing uses a CRISPR-associated (Cas) enzyme. Several Cas enzymes have been identified in various
bacterial systems. Some of these have been adapted for use in eukaryotic systems. The 2 most popular enzymes used in CRISPR genome editing are Cas9 and Cas12a (Cpf1), because they are highly functional, do not require binding to other enzymes, and
can be readily programmed to target the desired genomic DNA site. This programming occurs through a short guide RNA (gRNA) molecule that is designed by the researcher. The Cas9 or Cas12a enzyme binds the gRNA, forming a ribonucleoprotein (RNP), then, the gRNA directs the RNP
to the targeted site in the genomic DNA, where the Cas enzyme cuts the genome.
The IDT Alt-R CRISPR-Cas systems work as follows: You get chemically synthesized gRNA and recombinant, fully formed Cas enzyme (either Cas9 or Cas12a). You also can get positive and negative gRNA controls, an electroporation enhancer, a DNA donor
for homology-directed recombination (HDR), and an HDR enhancer. Once you have these reagents, you simply combine the gRNA and the Cas enzyme on the bench, forming the RNP, and then use electroporation to deliver this RNP to your cells. This is how genome
editing is accomplished quickly and accurately. Ultimately you can determine your results using next-generation sequencing (NGS).
What is the difference between CRISPR-Cas9 and CRISPR-Cas12a?
The Cas9 and Cas12a enzymes have some important differences. Firstly, Cas9 recognizes a different protospacer-adjacent motif (PAM) than Cas12a does (Table 1). Secondly, Cas9 in nature uses a two-part guide RNA (crRNA and tracrRNA)—although
for research purposes, these two parts are often fused as a single-guide RNA (sgRNA)—whereas Cas12a naturally uses just a one-part gRNA (crRNA).