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CRISPR editing is a brilliant and innovative adaptation of what nature already does well: nucleic acid base pair recognition, enzymatic cleavage, and DNA repair mechanisms. The editing process can be separated into two parts: cutting and repair. Both of these steps contain crucial elements for successful genome editing.
Separator

A molecular scalpel

The first step is to introduce a double-stranded DNA cut in the genomic region that has been targeted for a precise edit. The genomic cut is lethal to the cell, therefore requiring repair, which provides an opportunity to introduce the desired change. The specifics vary between organisms and systems, but the general method relies on a set of common principles, including the three essential CRISPR components: the nuclease, gRNA and PAM site.

Cas nuclease

First, the intended edit site is targeted for a double-strand break by a CRISPR-associated (Cas) nuclease. There are different types and variants of Cas nucleases with distinct features. 

First, the intended edit site is targeted for a double-strand break by a CRISPR-associated (Cas) nuclease. There are different types and variants of Cas nucleases with distinct features. The first one implemented for gene editing was Cas9, a Type II single-protein endonuclease. Inscripta’s MAD7 is a Type V Cas nuclease that belongs to the Cas12a family [1]. Among other differences, MAD7 DNA cleavage generates single-stranded overhangs, whereas Cas9 produces a blunt-end cut.

gRNA

The second essential component is the guide RNA (gRNA), which directs the Cas nuclease to the target site. The gRNA consists of a nucleotide sequence complementary to the target DNA, called a spacer, and a secondary structure, referred to as the handle. 

The second essential component is the guide RNA (gRNA), which directs the Cas nuclease to the target site. The gRNA consists of a nucleotide sequence complementary to the target DNA, called a spacer, and a secondary structure, referred to as the handle. While the unmodified Cas9 system requires a second molecule called tracrRNA to function, the Cas12a family gRNA is a single molecule and is slightly shorter. See the reference [2] for a comparison of the two systems. 

PAM site

Additionally, successful nuclease binding and activation requires the presence of a Protospacer Adjacent Motif (PAM) site in the targeted genomic region. The PAM is a short (typically 3 or 4 nucleotides), highly specific motif located either directly upstream or downstream of the gRNA spacer recognition site. 

Additionally, successful nuclease binding and activation requires the presence of a Protospacer Adjacent Motif (PAM) site in the targeted genomic region. The PAM is a short (typically 3 or 4 nucleotides), highly specific motif located either directly upstream or downstream of the gRNA spacer recognition site. PAM serves as a binding signal for the nucleases that initiates the gRNA pairing. The canonical PAM sequence for Cas9 is 5′-NGG‑3′, whereas MAD7 recognizes a T‑rich PAM.

Biochemical sutures

After the nuclease cuts the genomic DNA, the desired edit must be incorporated into the target site using DNA repair mechanisms. The homology-directed repair (HDR) pathway provides a high-fidelity method to introduce the change by providing a repair template with homology arms to the DNA flanking the double-strand break. The repair template also replaces the PAM site, preventing subsequent recognition of the same target sequence by CRISPR nucleases. The presence and activity of HDR pathways varies between organisms and often requires the heterologous expression of the HDR machinery.

References
  • [1] Zetsche, Bernd, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.” Cell 163.3 (2015): 759 – 771.
  • [2] Swarts, Daan C., and Martin Jinek. Cas9 versus Cas12a/​Cpf1: Structure – function comparisons and implications for genome editing.” Wiley Interdisciplinary Reviews: RNA 9.5 (2018): e1481.