Using CRISPR for Genome Editing
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR sequences were originally identified in the Escherichia coli (E. coli) genome, and were found to function as part of an RNA-based adaptive immune system to target and destroy genetic parasites at the DNA level.
CRISPR-associated protein (Cas) is an endonuclease that cuts foreign DNA, allowing integration into the host genome. Cleavage only occurs when there is a protospacer adjacent motif (PAM) around the targeted sequence of the invading DNA, ensuring highly accurate targeting. Researchers studying CRISPR have adapted it for use as a tool for genetic modification of the target host genome. CRISPR/Cas9 has recently become a popular genome editing tool, due to its simplicity and versatility.
Once Cas9 nucleases are guided to the target DNA and create a double strand break 3-4 bases upstream from the PAM sequences, there are two ways the double strand break (DSB) can be repaired. If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel that effectively knocks out protein function. Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.
Of the other gene editing technologies available, CRISPR/Cas9 has stood out for its simplicity and efficacy. The CRISPR system requires only a few simple DNA constructs to encode the gRNA and Cas9, and if knock-in is being performed, the donor template for HR. As a result, CRISPR gene editing is an approachable technique for use in any lab regardless of molecular biology expertise. The table below outlines a few of the key differences between CRISPR gene editing and other popular techniques.