Methods for simultaneous precise editing of multiple genes in human cells and screening of monoclonal cells

The discovery of CRISPR technology has brought hope for an efficient, reliable, and convenient genome editing tool

CRISPR nucleases and guide RNAs (gRNAs) are used to introduce double-strand breaks (DSBs) in the genome, and the accuracy and efficiency of genome editing is affected by cellular DSB repair pathways, including homology-directed repair (HDR), Non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), etc.

Stephan Riesenberg, a researcher at the Max Planck Institute for Evolutionary Anthropology in Germany, shared a CRISPR-Cas9-based multiplex genome editing using PRKDC gene mutation and kinase inhibitor M3814 to inhibit the catalytic subunit of DNA-dependent protein kinase (DNA- PKcs) activity, increasing the efficiency of gene editing HDR, making it possible to precisely edit up to 4 genes simultaneously in the same cell ^[2]

K3753R mutation of PRKDC gene in human cells promotes HDR

In order to improve the efficiency of gene editing targeting DSB and reduce the possibility of DSB off-target, a human induced pluripotent stem cell (hiPSC) cell line {cultured in a plate containing Matrigel Matrix (Corning, 35248), the medium is replaced with daily supplements ( StemCell Technologies, 05852) mTeSR1 medium (StemCell Technologies, 05851)}, doxycycline-inducible Cas9 (iCRISPR-Cas9n) carrying the D10A mutation to induce DNA single-strand breaks ^[3]

Design gRNAs and 14 single-stranded oligodeoxynucleotide donors (ssODNs), doxycycline-induced Cas9n expression, and transfect gRNAs and ssODNs

Figure 1: Inactivation of DNA-PKcs promotes HDR

The 3 genes with the lowest HDR frequencies in KR cells were selected and Cas9 was used instead of Cas9n, and the HDR of two genes increased from 35% and 20% to 73% and 87%, respectively (Fig.

Figure 2: HDR frequency changes in KR cell lines with different nucleases

To test whether the increase in HDR seen in hiPSCs was dependent on cell type or enzymes used, HEK293 cells {(ECACC, 85120602) were gene-edited in culture medium containing 10% fetal bovine serum (FBS) (SIGMA, F2442) and 1 %NEAA (SIGMA, M7145) Dulbecco's Modified Eagle's Medium/F-12 (Gibco, 31330-038)} and K562 cells {(ECACC, 89121407) in Iscove's Modified Dulbecco's Medium with 10% FBS ( ThermoFisher, 12440053)}, gene-edited monoclonal cells were sorted on a single-cell printer for library preparation and Illumina sequencing

Figure 3: Different DNA-PKcs K3753R cell lines can promote HDR

Precise genome editing of multiple genes simultaneously

Pairwise combinations of four different genes were electroporated into KR cells of iPSCs after induction of Cas9n expression

For triple editing, approximately one-third of sorted single-cell-derived clones (SCCs) carried all target genes (6 chromosomes) as homozygotes; 6% of quadruple-edited SCCs had 4 amino acids Replaced homozygous cell clones, 1000-fold higher than expected (Fig.

Figure 4: Multiplexed precise genome editing using inactivated DNA-PKcs cell lines

Genomic stability of cells expressing inactivated DNA-PKcs is not affected

To verify whether inactivation of the DNA-PKcs catalytic activity of 409B2-iCRISPR iPSCs results in genomic instability, using Trypsin-Giemsa Banding (GTG) reagent and spectral karyotyping (SKY) karyotyping, it was shown that a KR-KCS clone was observed in a Polyploid metaphase chromosomes (Fig.

To assess other types of genetic instability, DNA-PKcs were generated for WT, WT cells after 48 passages to generate KR clones, and KR cells after 24 passages to generate triple-edited clones KR-KSC (KATNAI-SLITRKI-CALDI editing) (Figure 5A).
) for whole-genome sequencing
.
Comparing 1mb of genome coverage along chromosomes, the only difference between KR and KR-KSC compared to WT was a decrease in chromosome 9 coverage, indicating a heterozygous deletion (Fig.
5E)
.
Whole-genome sequencing revealed a total of 19 novel heterozygous single nucleotide variants (SNVs) between KR and KR-KSC cells and 3 novel heterozygous SNVs in KR-KSC cells; no three cells were found What is the difference between indels between departments
.
According to the whole genome results, the mutation rate of WT cells was 21, and that of KR cells was 7, which proved that KR and KR-KSC did not increase the probability of mutation
.

Figure 5: Genomic stability of DNA-PKcs K3753R cells

Small molecule M3814 transiently inactivates DNA-PKcs

KR，，DNA-PKcs，DNA-PKcsHDR
。M3814DNA-PKcs，2 μMM38143，(Cytena)，DNAPCR，Illumina
。

，WT K562Cas9DSBHDR18%81%，，409B2 hiPSCsHDR(6A6B)
。DNA-PK，M3814HDR(6C)
。DNA，M3814M3814，，M3814，10%；M3814，76%
。，M3814HDR(6D6E)
。，K3753RM3814DNA-PKcs，PGE，
。

6：M3814DNA-PKcs

This article demonstrates that human cells can utilize the K3753R mutation of the PRKDC gene to improve the efficiency of PGE in gene editing, and can combine with the small molecule M3814 to further promote HDR
.
Under this approach, the K3753R mutation was not found to reduce genome stability; there were fewer translocations and fewer mutations per passage after bleomycin treatment
.
Damage to the error-prone NHEJ may cause cells to repair DSBs more frequently through the error-prone HDR, or cells may undergo apoptosis, causing surviving cells to maintain their genomes in a more accurate form
.
The DNA-PKcs inhibitor M3814 was able to increase HDR to a certain extent, which was comparable to PRKDC mutation
.
Permanent DNA-PKcs inactivation results in severe combined immunodeficiency in mammals due to inability to undergo V(D)J recombination, so M3814 could be used in gene therapy to improve HDR efficiency to achieve therapeutic goals
.

[1].
Xue C, Greene EC.
DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.
Trends Genet.
2021 Jul;37(7):639-656.
doi: 10.
1016/j.
tig.
2021.
02.
008.
Epub 2021 Apr 22.

[2].
Riesenberg S, Chintalapati M, Macak D, Kanis P, Maricic T, Pääbo S.
Simultaneous precise editing of multiple genes in human cells.
Nucleic Acids Res.
2019 Nov 4;47(19):e116.
doi: 10.
1093 /nar/gkz669.

[3].
Riesenberg S, Maricic T.
Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells.
Nat Commun.
2018 Jun 4;9(1):2164.
doi: 10.
1038/s41467-018-04609-7 .

As one of the greatest scientific achievements of the 21st century, gene editing has a wide range of applications
.
In order to obtain successfully gene-edited cell lines, the transfected cell pool needs to be enriched and cloned to create a unified monoclonal cell line, pending further characterization
.
Traditional sorting methods such as limiting dilution are time-consuming, labor-intensive, and inefficient; cells sorted by FACS are easily damaged by the pressure of sheath fluid, resulting in a low monoclonal rate
.
At the same time, both methods cannot provide proof of single-cell origin and cannot meet regulatory requirements
.

Cytena's single-cell printer uses patented inkjet printing technology to gently and efficiently sort single cells, simplifying the gene editing workflow and greatly improving the efficiency of single-cloning
.
As the general agent of Cytena's single-cell printer, Ai Beitai Biotechnology Co.
, Ltd.
provides you with the best pre-sale and after-sale service
.

Table 1: Single Cell Printer Parameters