CRISPR-Cas gene knock-in (KI) refers to a gene editing technology that inserts exogenous genes into the genome through homologous recombination (HDR) to enable them to be stably expressed in cells. The knocked-in exogenous gene can be a coding gene with protein expression function, a DNA element involved in gene regulation, or a non-functional DNA sequence.

Gene knock-in technology plays an important role in many aspects such as gene function research and disease treatment. However, in the actual operation process, it is not easy to obtain a homozygous knock-in cell line. Because the success of knock-in cell line construction is influenced by various factors, including the length of the knock-in gene fragment, homologous recombination efficiency, and cell line type, which can impact editing efficiency, this also makes the knock-in technology challenging. This article introduces the principles of constructing KI cell lines and the details that need to be paid attention to.

Principle of CRISPR-Cas gene knock-in

Under the guidance of sgRNA, Cas endonuclease cuts the DNA double strand at a fixed point to produce a DNA gap, and then the organism initiates the homologous recombination repair pathway. When there is a highly homologous DNA template (Donor DNA), the Donor DNA will be used as a template for repair, thereby achieving the purpose of introducing the target fragment into the genome.

In this process, the cutting efficiency of Cas, the probability of repairing the double-stranded DNA break in the cell by homologous recombination, and the probability of recombination with the Donor template together affect the final gene editing efficiency. This is also the reason for the low rate of positive clones.

Precautions for constructing knock-in cell lines

The construction process of knock-in cell lines is to first design sgRNA and Donor templates, and then transfer sgRNA, Cas protein and Donor template into cells through ribonucleoprotein (RNP) or plasmid methods. Taking the RNP method as an example, the synthesized sgRNA, purified Cas protein and Donor template are electroporated into the target cells, and resistance screening, PCR identification and sequencing are used to determine whether the knock-in cell line is successfully constructed. In this process, the design of sgRNA and Donor template will affect the final editing results. The following summarizes some of the reasons that affect gene editing efficiency:

1. Design of sgRNA

sgRNA-Cas in the knock-in gene editing system acts like a pair of scissors to precisely cut double-stranded DNA. The sharpness and accuracy of these scissors can directly affect the editing efficiency of the knock-in gene editing system. The design of sgRNA is an important step, which can affect the accuracy of insertion and cutting efficiency of the target fragment. Therefore, it is necessary to comprehensively consider multiple factors to scientifically design sgRNA. When designing, it is necessary to comprehensively consider the sequence, position, positive and negative strands, GC content, potential off-target sites and other information of the candidate editing site. The following principles can be used to design sgRNA:

(1) The cutting efficiency of the positive and negative strand targets of the candidate editing site is the same, so both targets can be designed;

(2) The GC content of the target should not be less than 40%. The target sequence with a higher GC content (50-70%) has a higher targeting efficiency;

(3) There should not be more than 4 consecutive T bases in the target to prevent it from becoming a transcription termination signal for RNA Pol III;

(4) In order to avoid or reduce non-specific off-target mutations, target specificity analysis should be performed. It is recommended to use the CCTop (https://cctop.cos.uni-heidelberg.de/) online website for prediction. Finally, select one or more sgRNAs with high cutting efficiency and good specificity to carry out the experiment.

2. Selection of RNP method and plasmid method

The presence of Cas protein and sgRNA in cells is an important step in achieving gene knock-in. Common methods for expressing Cas protein and sgRNA include ribonucleoprotein method (RNP) and plasmid method. The plasmid method has a relatively simple process, short cycle and low cost, but it cannot stably express Cas protein. The RNP method has a low off-target probability, no DNA integration risk and promoter compatibility issues, and can complete the cutting efficiency test in 5-6 days, significantly shortening the experimental cycle. Gene editing occurs quickly 12-24 hours after transfection, while plasmid vectors generally require 72 hours. Therefore, the RNP method has higher efficiency than plasmid expression vectors. And it does not introduce redundant expression elements or cause cytotoxicity.

3. Donor template design

Donor template has an important influence on the efficiency of homologous recombination. The following factors need to be considered during the experiment:

(1) The distance between the insertion site and the double-stranded DNA gap (DSB). The distance should not exceed 100 bp, and the ideal distance is within 10 bp. If it exceeds this distance, the recombination efficiency will be greatly reduced.

(2) The length of the knock-in fragment. ssODN can be used for the knock-in of short fragments (such as Tag tags, etc.). The knock-in of long fragments requires a dsDNA plasmid as a repair template. It should be noted that the knock-in fragment should not be too long, generally not exceeding 5 kb (including resistance and fluorescence screening). Too long will reduce the recombination efficiency.

(3) The length of the homology arm. Generally speaking, lengthening the homology arm can improve the recombination efficiency to a certain extent. And the larger the inserted fragment, the longer the homology arm length is required, and the homology arm is generally selected to be about 1500 bp.

(4) The selection of homology arm template. It is recommended that the homology arm be amplified using the target cell genome to be mutated as a template to avoid differences in the genomes of different cells leading to different homology arms and reduced recombination efficiency.

4. Cell type and growth state

Before transient transfection, attention should be paid to whether the cell type is appropriate, such as the proliferation rate of the cells. Cells that proliferate too slowly also have low recombination efficiency. It is also necessary to ensure that the cells are in a good growth state, in the logarithmic growth phase, at a suitable growth density, and free of mycoplasma contamination.

5. Transient transfection voltage

The appropriate transient transfection voltage can affect the editing efficiency and cell survival. Too low voltage will affect the editing efficiency, and too high voltage will cause cell death. Therefore, it is necessary to explore the appropriate transient transfection voltage for different cell types. Generally speaking, the transient transfection voltage is related to the diameter of the cell. The larger the diameter, the lower the voltage. It is recommended to combine the preliminary experiment to select the best transient transfection voltage.