PMID- 34972984
OWN - NLM
STAT- MEDLINE
DCOM- 20220331
LR  - 20231213
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 2445
DP  - 2022
TI  - CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human 
      Cancer Cells.
PG  - 39-50
LID - 10.1007/978-1-0716-2071-7_3 [doi]
AB  - Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent 
      protein degradation pathway. A critical molecular component of CMA is the 
      lysosome-associated membrane protein (LAMP) type 2A, which is required for 
      substrate uptake by the lysosome. Defects in the CMA pathway have been associated 
      with various human pathologies, including malignancies, increasing the overall 
      interest in methods to monitor this selective autophagy process. Yet isogenic 
      LAMP-2A knockout cancer cell models are still lacking. This is likely to depend 
      on challenges related to that human LAMP-2 gene undergoes alternative splicing of 
      its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. 
      However, without assessment of the impact of LAMP-2A loss of function 
      specifically in human cells, the involvement of CMA in human pathologies, 
      including carcinogenesis remains speculative. Here, we describe the generation of 
      isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, 
      without affecting the other two isoforms, allowing for experimental evaluation of 
      LAMP-2A, thus CMA in human cancer models.
CI  - (c) 2022. The Author(s), under exclusive license to Springer Science+Business 
      Media, LLC, part of Springer Nature.
FAU - Yu, Ting-Ting
AU  - Yu TT
AD  - Department of Physiology and Pharmacology, Solnavagen 9, Biomedicum, Karolinska 
      Institutet, Stockholm, Sweden.
AD  - Department of Medical Genetics, School of Basic Medical Science, Longmian Avenue 
      101, Nanjing Medical University, Nanjing, China.
FAU - Zhou, Xun
AU  - Zhou X
AD  - Department of Physiology and Pharmacology, Solnavagen 9, Biomedicum, Karolinska 
      Institutet, Stockholm, Sweden.
FAU - Vakifahmetoglu-Norberg, Helin
AU  - Vakifahmetoglu-Norberg H
AD  - Department of Physiology and Pharmacology, Solnavagen 9, Biomedicum, Karolinska 
      Institutet, Stockholm, Sweden. Helin.norberg@ki.se.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Lysosomal Membrane Proteins)
RN  - 0 (Protein Isoforms)
SB  - IM
MH  - Autophagy
MH  - CRISPR-Cas Systems
MH  - *Gene Editing
MH  - Humans
MH  - Lysosomal Membrane Proteins/genetics/metabolism
MH  - Lysosomes/metabolism
MH  - *Neoplasms/genetics/metabolism
MH  - Protein Isoforms/genetics/metabolism
OTO - NOTNLM
OT  - Autophagy
OT  - CRISPR-Cas9
OT  - Cancer
OT  - Chaperone-mediated autophagy
OT  - Gene editing
OT  - LAMP-2A
EDAT- 2022/01/02 06:00
MHDA- 2022/04/01 06:00
CRDT- 2022/01/01 05:27
PHST- 2022/01/01 05:27 [entrez]
PHST- 2022/01/02 06:00 [pubmed]
PHST- 2022/04/01 06:00 [medline]
AID - 10.1007/978-1-0716-2071-7_3 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2022;2445:39-50. doi: 10.1007/978-1-0716-2071-7_3.