Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in<i>Mycobacterium abscessus</i>
ABSTRACT Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, poses a significant public health challenge because of its ability to cause many types of serious infections in humans. While genetic manipulation tools forM. abscessusare still being developed, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems have shown promise in generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy inM. abscessus. Moreover, our research uncovered an unexpected finding: contrary to previous observations inMycobacterium tuberculosisand other species, the inhibition of RecA or disruption of key genes in the homologous recombination or single-strand annealing pathways resulted in a significant decrease in NHEJ repair efficiency inM. abscessus. This discovery challenges the established perspectives and offers new insights into the interaction among the three DSB repair pathways in Mycobacterium species.IMPORTANCE There are still very few genetic manipulation tools available forMycobacterium abscessus.. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing inM. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption of either HR or single-strand annealing (SSA) DNA double-strand breaks (DSBs) repair pathways not only failed to enhance but also significantly reduced the gene editing efficiency inM. abscessus. This suggests that NHEJ repair inM. abscessusmay require components from both HR and SSA pathways, highlighting a complex interaction among the DSB repair pathways inM. abscessus..
| Medienart: |
|
|---|
Preprint| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 01. Feb. Zur Gesamtaufnahme - year:2024 |
|---|
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Zeng, Sanshan [VerfasserIn] |
|---|
| Links: |
Volltext [kostenfrei] |
|---|
| Themen: |
|---|
| doi: |
10.1101/2024.01.29.577284 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
XBI042349842 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | XBI042349842 | ||
| 003 | DE-627 | ||
| 005 | 20240202092125.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 240201s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1101/2024.01.29.577284 |2 doi | |
| 035 | |a (DE-627)XBI042349842 | ||
| 035 | |a (biorXiv)10.1101/2024.01.29.577284 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Zeng, Sanshan |e verfasserin |0 (orcid)0009-0002-2047-7669 |4 aut | |
| 245 | 1 | 0 | |a Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in<i>Mycobacterium abscessus</i> |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a Computermedien |b c |2 rdamedia | ||
| 338 | |a Online-Ressource |b cr |2 rdacarrier | ||
| 520 | |a ABSTRACT Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, poses a significant public health challenge because of its ability to cause many types of serious infections in humans. While genetic manipulation tools forM. abscessusare still being developed, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems have shown promise in generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy inM. abscessus. Moreover, our research uncovered an unexpected finding: contrary to previous observations inMycobacterium tuberculosisand other species, the inhibition of RecA or disruption of key genes in the homologous recombination or single-strand annealing pathways resulted in a significant decrease in NHEJ repair efficiency inM. abscessus. This discovery challenges the established perspectives and offers new insights into the interaction among the three DSB repair pathways in Mycobacterium species.IMPORTANCE There are still very few genetic manipulation tools available forMycobacterium abscessus.. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing inM. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption of either HR or single-strand annealing (SSA) DNA double-strand breaks (DSBs) repair pathways not only failed to enhance but also significantly reduced the gene editing efficiency inM. abscessus. This suggests that NHEJ repair inM. abscessusmay require components from both HR and SSA pathways, highlighting a complex interaction among the DSB repair pathways inM. abscessus. | ||
| 650 | 4 | |a Biology |7 (dpeaa)DE-84 | |
| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a Ju, Yanan |4 aut | |
| 700 | 1 | |a Alam, Md Shah |4 aut | |
| 700 | 1 | |a Lu, Ziwen |4 aut | |
| 700 | 1 | |a Hameed, H.M. Adnan |4 aut | |
| 700 | 1 | |a Li, Lijie |4 aut | |
| 700 | 1 | |a Tian, Xirong |4 aut | |
| 700 | 1 | |a Fang, Cuiting |4 aut | |
| 700 | 1 | |a Fang, Xiange |4 aut | |
| 700 | 1 | |a Ding, Jie |4 aut | |
| 700 | 1 | |a Wang, Xinyue |4 aut | |
| 700 | 1 | |a Hu, Jinxing |4 aut | |
| 700 | 1 | |a Wang, Shuai |4 aut | |
| 700 | 1 | |a Zhang, Tianyu |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t bioRxiv.org |g (2024) vom: 01. Feb. |
| 773 | 1 | 8 | |g year:2024 |g day:01 |g month:02 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1101/2024.01.29.577284 |z kostenfrei |3 Volltext |
| 912 | |a GBV_XBI | ||
| 951 | |a AR | ||
| 952 | |j 2024 |b 01 |c 02 | ||