Reference SummaryLara-Saez I, Proc Natl Acad Sci U S A 2024 Jul 9;121(28):e2322917121

Title

 Nonviral CRISPR/Cas9 mutagenesis for streamlined generation of mouse lung cancer models.

Authors

 Lara-Saez I; Mencia A; Recuero E; Li Y; Garcia M; Oteo M; Gallego MI; Enguita AB; de Prado-Verdun D; A S; Wang W; Garcia-Escudero R; Murillas R; Santos M

Journal

 Proc Natl Acad Sci U S A

Volume

 121

Issue

 28

Year

 2024

Pages

 e2322917121

Abstract

 Functional analysis in mouse models is necessary to establish the involvement of a set of genetic variations in tumor development. A modeling platform to facilitate and cost-effectively analyze the role of multiple genes in carcinogenesis would be valuable. Here, we present an innovative strategy for lung mutagenesis using CRISPR/Cas9 ribonucleoproteins delivered via cationic polymers. This approach allows the simultaneous inactivation of multiple genes. We validate the effectiveness of this system by targeting a group of tumor suppressor genes, specifically Rb1, Rbl1, Pten, and Trp53, which were chosen for their potential to cause lung tumors, namely small cell lung carcinoma (SCLC). Tumors with histologic and transcriptomic features of human SCLC emerged after intratracheal administration of CRISPR/polymer nanoparticles. These tumors carried loss-of-function mutations in all four tumor suppressor genes at the targeted positions. These findings were reproduced in two different pure genetic backgrounds. We provide a proof of principle for simplified modeling of lung tumorigenesis to facilitate functional testing of potential cancer-related genes.

Links

 J:351397 – MGI References
38959035 – National Library of Medicine/PubMed

Models
Strain Model Name Treatment Agent(s) Organ Affected Frequency Model Details
NMRI-Foxn1nu Lung carcinoma - large cell neuroendocrine
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - large cell neuroendocrine
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

observed - 7
NMRI-Foxn1nu Lung carcinoma - large cell neuroendocrine
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0 - observed
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - large cell neuroendocrine
  • Qfrt Y4 L5-Cationic polymer Y4 L5 carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0 - observed
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - large cell neuroendocrine
  • Qfrt Y4Cys-Cationic polymer Y4Cys carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0
NMRI-Foxn1nu Lung carcinoma - large cell neuroendocrine
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

22
NMRI-Foxn1nu Lung carcinoma - small cell lung (SCLC)
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

observed - 75
NMRI-Foxn1nu Lung carcinoma - small cell lung (SCLC)
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

observed - 22
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - small cell lung (SCLC)
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

observed - 47
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - small cell lung (SCLC)
  • Qfrt Y4 L5-Cationic polymer Y4 L5 carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

observed - 10
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - small cell lung (SCLC)
  • Qfrt Y4Cys-Cationic polymer Y4Cys carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0 - 20
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung carcinoma - small cell lung (SCLC)
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lymph node

7
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung hyperplasia - neuroendocrine
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

observed - 7
NMRI-Foxn1nu Lung hyperplasia - neuroendocrine
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung hyperplasia - neuroendocrine Lung

0
NMRI-Foxn1nu Lung hyperplasia - neuroendocrine
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lung

observed - 10
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung hyperplasia - neuroendocrine
  • Qfrt Y4Cys-Cationic polymer Y4Cys carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Lung hyperplasia - neuroendocrine
  • Qfrt Y4 L5-Cationic polymer Y4 L5 carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Lung

0
NMRI-Foxn1nu Trachea carcinoma
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Trachea

22
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Trachea carcinoma - small cell lung (SCLC)
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Trachea

observed - 60
NMRI-Foxn1nu Trachea carcinoma - small cell lung (SCLC)
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Trachea

observed - 56
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Trachea carcinoma - small cell lung (SCLC) Trachea

observed
NMRI-Foxn1nu Trachea carcinoma - small cell lung (SCLC)
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Trachea

observed
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Trachea carcinoma - small cell lung (SCLC)
  • Qfrt Y4 L5-Cationic polymer Y4 L5 carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Trachea

30
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen Trachea carcinoma - small cell lung (SCLC)
  • Qfrt Y4Cys-Cationic polymer Y4Cys carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 Trachea

20
NMRI-Foxn1nu (Unspecified organ) carcinoma - large cell neuroendocrine
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 (Unspecified organ)

25
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Qfrt Y4 Linear-Cationic polymer Y4 Linear carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 (Unspecified organ)

67
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Qfrt Y4 L5-Cationic polymer Y4 L5 carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 (Unspecified organ)

0 - 30
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Qfrt Y4Cys-Cationic polymer Y4Cys carrying RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 (Unspecified organ)

0 - 20
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • frt Y4Linear-Cationic polymer Y4Linear carrying RNAP guide for frt.
 (Unspecified organ)

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • frt Y4L5-Cationic polymer Y4L5 carrying RNAP guide for frt.
 (Unspecified organ)

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Y4Linear-Cationic polymer Y4Linear
 (Unspecified organ)

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Y4L5-Cationic polymer Y4L5
 (Unspecified organ)

0
B6.Cg-Gt(ROSA)26Sortm1.3(CAG-tdTomato,-EGFP)Pjen (Unspecified organ) tumor
  • Qfrt-RNAP guides for Rb, Rb1l, Pten, Trp53 and frt
 (Unspecified organ)

0
FVB (Unspecified organ) tumor
  • Q-RNAP guides for Rb, Rb1l, Pten, Trp53
 (Unspecified organ)

0
NMRI-Foxn1nu (Unspecified organ) tumor
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 (Unspecified organ)

44 - 75
NMRI-Foxn1nu (Unspecified organ) tumor
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 (Unspecified organ)

10 - 22
NMRI-Foxn1nu (Unspecified organ) tumor
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Liver

33
NMRI-Foxn1nu (Unspecified organ) tumor
  • Q Y4Linear-Cationic polymer Y4Linear carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Lymph node

10
NMRI-Foxn1nu (Unspecified organ) tumor
  • Q Y4L5-Cationic polymer Y4L5 carrying RNAP guides for Rb, Rb1l, Pten, and Trp53.
 Liver

10