CLL-384: Proliferation and Unmutated IGHV Status are Associated with Homologous Recombination Activity in CLL Cells

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

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Homologous recombination between coinjected DNA sequences peaks in early to mid-S phase

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2294-2295.1987 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2294-2295

Author(s):

E A Wong ◽

M R Capecchi

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

Dna Sequences ◽

S Phase ◽

Recombination Activity ◽

Rat Fibroblasts

We have examined the effect of cell cycle position on homologous recombination between plasmid molecules coinjected into synchronized rat fibroblasts. Recombination activity was found to be low in G1 and to rise 10- to 15-fold, peaking in early to mid-S phase.

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Elevated Homologous Recombination Activity in Fanconi Anemia Fibroblasts

Journal of Biological Chemistry ◽

10.1074/jbc.272.37.23328 ◽

1997 ◽

Vol 272 (37) ◽

pp. 23328-23333 ◽

Cited By ~ 37

Author(s):

Bhaskar Thyagarajan ◽

Colin Campbell

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Recombination Activity

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Thymidylate stress induces homologous recombination activity in mammalian cells

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/0027-5107(91)90124-7 ◽

1991 ◽

Vol 246 (1) ◽

pp. 215-220 ◽

Cited By ~ 10

Author(s):

Mishina Yuji ◽

Ayusawa Dai ◽

Seno Takeshi ◽

Hideki Koyama

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Recombination Activity

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Homologous and illegitimate recombination in developing Xenopus oocytes and eggs

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6897-6906.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6897-6906

Author(s):

C W Lehman ◽

M Clemens ◽

D K Worthylake ◽

J K Trautman ◽

D Carroll

Keyword(s):

Homologous Recombination ◽

Developmental Stages ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Xenopus Laevis Oocytes ◽

Recombination Activity ◽

Exogenous Dna ◽

Strand Breaks ◽

Nuclear Extracts ◽

Reaction Proceeds

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

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Homologous recombination between coinjected DNA sequences peaks in early to mid-S phase.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2294 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2294-2295 ◽

Cited By ~ 50

Author(s):

E A Wong ◽

M R Capecchi

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

Dna Sequences ◽

S Phase ◽

Recombination Activity ◽

Rat Fibroblasts

We have examined the effect of cell cycle position on homologous recombination between plasmid molecules coinjected into synchronized rat fibroblasts. Recombination activity was found to be low in G1 and to rise 10- to 15-fold, peaking in early to mid-S phase.

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A recA Null Mutation May Be Generated in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.00951-06 ◽

2006 ◽

Vol 188 (19) ◽

pp. 6771-6779 ◽

Cited By ~ 16

Author(s):

Tzu-Wen Huang ◽

Carton W. Chen

Keyword(s):

Homologous Recombination ◽

Streptomyces Coelicolor ◽

Sos Response ◽

Gene Replacement ◽

Repair Pathway ◽

Wild Type ◽

Recombination Activity ◽

Almost All ◽

First Time ◽

Null Mutants

ABSTRACT The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.

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Phosphoregulation of Rad51/Rad52 by CDK1 functions as a molecular switch for cell cycle–specific activation of homologous recombination

Science Advances ◽

10.1126/sciadv.aay2669 ◽

2020 ◽

Vol 6 (6) ◽

pp. eaay2669 ◽

Cited By ~ 2

Author(s):

Gyubum Lim ◽

Yeonji Chang ◽

Won-Ki Huh

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

Kinase Activity ◽

Regulatory Mechanism ◽

Specific Cell ◽

Cyclin Dependent Kinase ◽

Recombination Activity ◽

M Phase ◽

Dna Binding Affinity ◽

Cycle Dependent

Homologous recombination is exquisitely activated only during specific cell phases. In the G1 phase, homologous recombination activity is completely suppressed. According to previous reports, the activation of homologous recombination during specific cell phases depends on the kinase activity of cyclin-dependent kinase 1 (CDK1). However, the precise regulatory mechanism and target substrates of CDK1 for this regulation have not been completely determined. Here, we report that the budding yeast CDK1, Cdc28, phosphorylates the major homologous recombination regulators Rad51 and Rad52. This phosphorylation occurs in the G2/M phase by Cdc28 in combination with G2/M phase cyclins. Nonphosphorylatable mutations in Rad51 and Rad52 impair the DNA binding affinity of Rad51 and the affinity between Rad52 rings that leads to their interaction. Collectively, our data provide detailed insights into the regulatory mechanism of cell cycle–dependent homologous recombination activation in eukaryotic cells.

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