CRISPR-Cas9 genome editing is changing the landscape of genomic research due to its ease-of-use and ability to create double-stranded breaks (DSBs) at desired genomic locations. So far, the most significant success of CRISPR technology has been obtained from use in site-specific disruption of genes for biomedical research. However, CRISPR-Cas9 genome editing can be extended beyond simple gene knock-out. The ability to precisely introduce an exogenous DNA fragment is of enormous value for researchers aiming to understand gene function or develop animal models for disease-causing mutations. This research will eventually serve as a gateway to repairing disease-causing mutations in clinical settings and altering genomes to increase yields of agricultural plants and animals.
CRISPR-Cas9–mediated HDR is desired but challenging
Once the double-stranded break is introduced by the CRISPR system, the cellular DNA repair mechanism follows one of two pathways: 1) non-homologous end joining (NHEJ), where broken ends of the DNA are joined together or 2) homology-directed repair (HDR), where cells use a DNA template, provided along with CRISPR components, to repair the DNA break via homologous recombination. NHEJ is very efficient but is prone to making mistakes, such as introducing deletions or insertions at the breakpoint. On the other hand, HDR generates precise changes but is inherently less efficient.
A unique solution to make your HDR experiment a success
To increase HDR efficiency, our scientists conducted extensive research on the modulation of HDR within the context of CRISPR-Cas9 mediated genome editing. Those efforts led to the development of a small molecule reagent that effectively diverts repair pathway towards HDR, successfully enhancing overall HDR efficiency. We tested the effectiveness of Alt-R HDR Enhancer in CRISPR editing experiments in 4 commonly used human cell lines (Jurkat, HEK-293, HeLa, and K562). IDT Ultramer oligonucleotides containing an EcoRI recognition site was designed as an HDR template, so that HDR efficiency could be assessed by determining the percent EcoRI digestion following PCR amplification of the edited region. As shown in Figure 1, Alt-R HDR Enhancer improved the HDR insertion rate in all 4 cell lines, without incurring discernible phenotypic change or cytotoxicity.
Figure 1. Alt-R HDR Enhancer improves HDR efficiency across multiple cell types. 4 µM of RNP complex (Alt-R S.p. Cas9 Nuclease V3 complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA) targeting human HPRT1 was delivered into multiple human cell lines via electroporation using the Amaxa® Nucleofector® System (Lonza), along with 4 µM of Alt-R Cas9 Electroporation Enhancer and 3 µM of Ultramer oligonucleotides as HDR template. After electroporation, cells were plated in media that contained either 30 µM of Alt-R HDR Enhancer (gray bar) or the same volume of DMSO as the negative control (light blue bar). The HDR template contains a 6-base Eco RI recognition site that enables assessment of HDR efficiency by restriction fragment length polymorphism (RFLP). Genomic DNA was isolated 48 hr (HEK-293 and HeLa) or 72 hr (Jurkat and K562) after electroporation, and the target locus was amplified by PCR. Amplicons were digested with EcoRI enzyme to determine the rate of HDR insertion. HDR = homology-directed repair; DMSO = dimethyl sulfoxide.
We also tested whether the improvement we observed with HDR enhancer is enzyme dependent. We compared both Streptococcus pyogenes Cas9 and Acidaminococcus sp. BV3L6 Cas12a (also known as Cpf1) and targeted multiple genomic locations using Jurkat cells. As shown in Figure 2, a significant HDR enhancement was observed across a variety of genomic locations, regardless of whether S.p. Cas9 or A.s. Cas12a was used to introduce DSBs.
Figure 2. Alt-R HDR Enhancer improves HDR mediated by S.p. Cas9 and A.s. Cas12a. Left: 4 µM of Alt-R Cas9 RNP targeting multiple sites in the human genome were delivered into Jurkat cells via electroporation using the Neon® Transfection System (Thermo Fisher), along with 4 µM of Alt-R Cas9 Electroporation Enhancer and 3 µM of IDT Ultramer oligos as HDR template. Right: 5 µM of Alt-R Cas12a RNP, together with 3 µM of Alt-R Cas12a Electroporation Enhancer and 3 µM of single-stranded DNA template were electroporated into Jurkat cells. After electroporation cells were plated in media that contained 30 µM of Alt-R HDR Enhancer (gray bar) or equal volume of DMSO as the negative control (light blue bar). A “no treatment” control group with no HDR enhancer (dark blue bar) was also included in the experiment. Genomic DNA was isolated 48–72 hr after electroporation, and HDR efficiency was assessed by EcoRI cleavage of the PCR products amplified from the target locus.
The proprietary Alt-R HDR Enhancer is available for maximizing your HDR potential. The HDR enhancer is shipped at a high concentration (3 mM) in a ready-to-use solvent (DMSO). While the efficiency of HDR and the relative improvement provided by the HDR enhancer may vary by cell line and sequence context, we offer a stepwise HDR protocol for maximizing your HDR efficiency while minimizing adverse effects often associated with the delivery procedure. To learn more about our HDR Enhancer, visit our Alt-R HDR Enhancer product page.