CRISPR Knock-In: A Guide

Introduction

Clustered regularly interspaced short palindromic repeat based (CRISPR-based) gene editing has revolutionized genetic engineering, with the possibility for the repair of damaged DNA lesions that cause rare disorders and cancer. Cas9 nuclease binds to a DNA site complementary to a guide RNA (gRNA) and creates strand breaks in a target genome site to reverse the detrimental effects of initial DNA mutations or damage. DNA is repaired to include nucleotides or sequences that create a normal genotype. While RNA-based gene silencing methods, such as RNA interference (RNAi), introduce transient changes in transcription and translation, CRISPR gene editing alters the genome irreversibly. In therapeutic applications, it can be valuable in correcting somatic mutations underlying rare genetic disorders and cancer. CRISPR-Cas9 gene editing is also used in research areas such as functional genomics, where knockout and knock-in studies can reveal insight into the significance of genes in cellular functions and disease onset.

CRISPR Knockout vs. Knock-in

The CRISPR-Cas9 systems edit the host genome in two ways. In knockout mutations, the targeted gene is removed, and the resulting double-stranded break is closed off by the innate cell DNA repair mechanisms, such as non-homologous end joining (NHEJ). The specific gene sequence is disrupted, and a loss of gene function is observed with the gene “knocked out.”

In contrast, knock-in mutations involve the incorporation of exogenous DNA to activate the homology-directed repair (HDR) pathway to correct the impairment or deficiency causing the disease. The exogenous DNA serves as a template to replace the deficient gene with high precision.

Mechanism for CRISPR/Cas9 Knock-in

CRISPR knock-in aims to direct homology-directed repair (HDR) toward double-stranded breaks (DSB) in the cell genome. The HDR pathway is a precise method of DNA repair, where a homologous DNA sequence serves as a template to induce desired changes at the DSB site.

Cells could employ HDR mechanisms to repair DSBs caused by several factors, from intracellular reactive oxygen species (ROS) to radiation and ultraviolet light¹. More importantly, CRISPR-Cas9 knock-in technology can stimulate HDR by providing donor DNA to direct HDR alongside the gRNA and Cas9 complex.

The main hindrance with HDR-based repair is that it must compete with NHEJ, which occurs more frequently than HDR. NHEJ quickly repairs double-stranded DNA breaks, but is less precise compared to HDR which uses a DNA template as well as protein complexes necessary for DNA end resection². That's why care must be given when designing knock-in editing to ensure that the cell is prompted to employ HDR rather than NHEJ.

Considerations for CRISPR Knock-In

The Ideal Donor DNA Template Type

The correct design of donor DNA is critical for administering desired mutations.

Double-stranded DNA (dsDNA) and short single-stranded DNA (ssDNA) are commonly used as repair templates. The correct template type depends on several factors, such as the number of base pairs the intended edit requires. ssDNA templates are suitable for carrying 50-100 base pairs of homology arms, while dsDNA is the choice for larger knock-ins of several hundred kb³. Both template types have advantages and disadvantages:

• dsDNA harbors a larger cargo capacity than ssDNA, allowing researchers to add tags or reporters that can help track the success of the editing⁴.
• ssDNA templates are more precise and less likely to cause cytotoxicity than dsDNA templates⁵.

In general, circular plasmid-based templates that integrate the edit sequence into a plasmid backbone were found to have low HDR efficiency⁶. Research shows that efficiency can be increased by designing self-cleaving plasmids that automatically release the edit sequence from the backbone⁷.

Methods for Increasing HDR Efficiency

HDR Activation and NHEJ Inhibition

One strategy for increasing HDR efficiency has been to increase HDR frequency relative to NHEJ by upregulating the HDR pathway. Song et al. used HDR enhancers to observe a 2-5-fold increase in knock-in efficiency⁸. A similar approach involved using small molecules to inhibit the NHEJ pathway, which yielded a substantial efficiency increase⁹. A group of studies focused on inhibiting another random repair mechanism called DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) alongside NHEJ to improve HDR-mediated DNA repair further¹⁰. Despite its innovative value in CRISPR knock-in, NHEJ inhibition comes with the caveat that it may interfere with the innate DSB repair mechanism. Because NHEJ is a prominent pathway maintaining genome stability, its inhibition may lead to significant side effects¹¹. Furthermore, more in vivo and ex vivo research is necessary to demonstrate the utility of these methods in gene therapy applications¹².

HDR efficiency can also be improved by increasing the fraction of the donor DNA that ends up at the DSB site. Efficiency was significantly increased in a group of studies by chemical modifications that tethered the donor DNA to the CRISPR-Cas9 complex¹³'¹⁴.

Improving CRISPR-Cas9 Delivery

Efficient delivery of the gRNA and Cas9 is essential for successful CRISPR knockout and knock-in. While plasmids can be preferable due to the simplicity of their design, they are prone to off-target delivery of the CRISPR agents and the template DNA.

In contrast, AAV vectors attracted attention for their robust HDR-activating properties, cell-type specificity, and low immunogenicity¹⁵; however, AAV-based CRISPR gene editing was found to drive prolonged Cas9 expression and previously associated with a p53-mediated DNA damage response that could cause adverse effects¹⁶.

RNA-based delivery of CRISPR-Cas9 offers advantages over viral vectors, such as smaller size and rapid and precise editing¹⁷. In that regard, lipid nanoparticles and retrovirus-like proteins are under investigation¹⁸.

Finally, CRISPR ribonucleoprotein (RNP) delivery emerged as a promising method. RNP delivery bypasses the complications with plasmids and viral vectors, as it already contains the Cas9 protein complexed to the targeting gRNA at the time of delivery. RNPs have reliable safety profiles, as the RNP constituents are rapidly degraded and discarded after genome editing. In other words, off-target effects and random genome integrations have shown to be significantly reduced compared to other methods¹⁹.

Applications of CRISPR Knock-in Protocols

CRISPR Knock-in protocols lend themselves to several applications in biotechnology.

For gene therapies, the precise insertion of a therapeutic gene is crucial to reversing deleterious mutations. CRISPR-mediated HDR corrects mutations permanently and can be applied in vivo or ex vivo. Ex vivo treatments involve the extraction of mutated cells from the patient, HDR-mediated correction, cell culture expansion of the corrected cells and re-administration into the patient. In vitro and in vivo studies are ongoing for the treatment of rare genetic diseases²⁰'²¹ and designing chimeric antigen receptor (CAR)-T cells for cancer treatment²²'²³.

Disease research can significantly benefit from CRISPR knock-in protocols to generate disease models for further research. Researchers can exploit HDR to induce disease-specific mutations. This method has been widely used to study cancer²⁴, Alzheimer's²⁵, and Huntington's diseases²⁶.

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