Kedu ka CRISPR/Cas9 si na-agbanwe ọgwụ, site na mgbochi nje gaa na ọgwụgwọ cell nke ọma.

N'ọgwụgwụ narị afọ nke 20, ndị ọkà mmụta sayensị na-amụ banyere mkpụrụ ndụ ihe nketa nje chọpụtara usoro usoro ugboro ugboro na-adịghị ahụkebe - e mesịrị kpọọ CRISPR. Na mbụ, a maghị ọrụ ha. Otú ọ dị, ka oge na-aga, nchọpụta gosiri na usoro ndị a bụ akụkụ nke "usoro nchekwa nchekwa ihe" nke nje bacteria na-eji echebe megide nje virus.

Mgbe nje butere nje bacteria, nje ahụ na-ewepụta iberibe DNA nke viral wee "dobe" ha n'ime usoro CRISPR ya. Ọ bụrụ na otu ụdị nje ahụ wakporo ọzọ, nje ahụ nwere ike ịmata ya site na usoro ezubere iche ma bepụ DNA viral.^[1].

Protein na-ahụ maka ọrụ ''molecular scissors'' bụ Cas9. Nchọpụta a mere ka ndị ọkà mmụta sayensị ghọta na ọ bụrụ na a ga-emepụta "usoro nlebara anya" ahụ n'ụzọ aka aka, CRISPR/Cas9 nwere ike gbanwee ka ọ bụrụ mkpụrụ ndụ ihe n'usoro - ngwa ndezi.

4. Isi usoro nke Gene edezi: kedu ka CRISPR/Cas9 si “ebipụ”?

Usoro ndezi mkpụrụ ndụ CRISPR - Cas9 nwere isi ihe abụọ:

• gRNA (nduzi RNA): onye na-ahụ maka “ichedo” site n'ịmata usoro DNA akọwapụtara
• Protein Cas9: na-ahụ maka “ịcha” site na iwebata njiri okpukpu abụọ - eriri na DNA

Enwere ike ịghọta usoro ọrụ ya bụ isi na usoro atọ:

Enwere ụzọ nrụzi isi abụọ:

• NHEJ (Na-abụghị - Njikọ Njikọ Homologous): na-ewebata ntinye ma ọ bụ nhichapụ, na-eduga n'ọgbagha mkpụrụ ndụ ihe nketa (knockout)
• HDR (Homology - Ndozi a na-eduzi): na-enyere aka ngbanwe ma ọ bụ ntinye kpọmkwem (kụọ - na)

Usoro a bụ ihe na-eme Cas9 ka ọ bụrụ otu n'ime mkpụrụ ndụ kacha arụ ọrụ nke ọma na nke dị iche iche - ngwaọrụ ndezi dị taa.

3. Site na bench ruo n'akụkụ akwa: Ngwa CRISPR/Cas9 na ọgwụgwọ cell
4. 1) CAR

Usoro ọgwụgwọ CAR -T na-agụnyekarị ikewapụ mkpụrụ ndụ T n'ebe onye ọrịa nọ, na-agbanwe ha site na mkpụrụ ndụ ihe nketaex vivo, wee nyeghachi ha azụ n'ime ahụ. Usoro a abụghị naanị oge - na-eri mana ọ dịkwa oke ọnụ, na-amachi nnukwu ngwa - oke ya.

Site na iwebata CRISPR - Teknụzụ Cas9, Otú ọ dị, ụzọ e si emepụta mkpụrụ ndụ CAR-T na-enwe mgbanwe dị ịrịba ama, na-eme ka ọgwụgwọ "ụwa niile" CAR-T na-esiwanye ike. Site na ndezi mkpụrụ ndụ ihe nketa nke mkpụrụ ndụ T, enwere ike ịkụtu mkpụrụ ndụ TCR iji gbochie graft - versus - ọrịa nnabata (GVHD). N'otu oge ahụ, ịkụpụ mkpụrụ ndụ ihe nketa metụtara ihe mgbochi ọrịa dịka A2AR nwere ike imeziwanye nnọgidesi ike na nnabata nke mkpụrụ ndụ CAR - T na ntọala ụlọ ọgwụ.^[2].

Na mgbakwunye, saịtị - ntinye a kapịrị ọnụ nke ihe owuwu CAR nwere ike ime ka mkpụrụ ndụ nwee ike ịmata na igbu antigens etuto. N'ihi nke a, ọgwụgwọ CAR-T na-amalite site na ụzọ ahaziri onwe ya zuru oke gaa na usoro ọgwụgwọ cellular "off-the-shelf".

2）Gene - Usoro ọgwụgwọ mkpụrụ ndụ edeziri: enwere ike gafere ọrịa kansa

CRISPR-Cas9 egosipụtala nnukwu nkwa ọ bụghị naanị na ọrịa immunotherapy nke ọrịa kansa kamakwa ọ na-agbasa ngwa ngwa n'ịgwọ ọrịa dị iche iche nke mkpụrụ ndụ ihe nketa na ọrịa na-efe efe. Dịka ọmụmaatụ, n'ọnọdụ ndị dị ka ọrịa sickle cell na β-thalassaemia, ndị nchọpụta na-edezi mkpụrụ ndụ hematopoietic stem cell iji weghachi okwu hemoglobin nkịtị, si otú a na-eleba anya na ọrịa ahụ na isi mmalite ya na imeziwanye nsonaazụ ụlọ ọgwụ.

Ugbu a, ụzọ nnyefe dị ka viral vectors, lipid nanoparticles (LNPs), na nje - dị ka ụmụ irighiri ihe (VLPs) ka a na-ejikarị eme usoro ọgwụgwọ vivo gene editing [3].

N'ozuzu, isi ụkpụrụ nke ụzọ ndị a bụ imezi "ọrịa-na-akpata mkpụrụ ndụ ihe nketa," kama ibelata mgbaàmà.

4. Ezigbo - Ọnọdụ ụwa: Nkuchi ahịa na-aga n'ihu

Usoro ọgwụgwọ CAR-T enwetala nnukwu ọganiihu na omume ụlọọgwụ ma na-aghọkwa usoro dị mkpa maka ọgwụgwọ ọrịa hematologic. Dịka ọmụmaatụ, Kymriah, ngwaahịa CAR-T nke mbụ akwadoro, enyerela mgbaghara ogologo oge na ụfọdụ ndị ọrịa nwere nnukwu ọrịa leukemia lymphoblastic. Yescarta sochiri nke a, bụ nke gosipụtara ọnụego nzaghachi zuru oke na nnukwu B-cell lymphoma, na-akwadokwa uru ụlọ ọgwụ nke CAR-T ọgwụgwọ. Ka ọ dị ugbu a, Carvykti, ezubere iche maka BCMA, emela nke ọma ma ịdị omimi na ịdịte aka nke nzaghachi na ndị ọrịa nwere otutu myeloma.

Ihe ịga nke ọma n'ụwa ndị a n'ezie na-egosi na ọgwụgwọ CAR-T na-emegharị usoro ọgwụgwọ ọrịa kansa. N'otu oge ahụ, njikọta nke CRISPR - Teknụzụ Cas9 na-akwọ ụgbọ ala CAR-T site na "nhazi ahaziri iche" gaa n'ichepụta "engineered." Site n'ịkụchapụ mkpụrụ ndụ ihe nketa dị ka TCR na PD-1, ọ ga-ekwe omume ibelata ọjụjụ na-alụso ọrịa ọgụ ma na-akwalite ọrụ antitumor. Nke a na-enyekwa nkwado teknụzụ dị oke egwu maka mmepe nke ngwaahịa sel zuru ụwa ọnụ, na-aga n'ihu na usoro ọgwụgwọ cell na nhazi ọkwa na scalability.

CRISPR - Cas9 esiwo na “ngwa nyocha” pụta wee bụrụ “usoro mmepe ọgwụ” siri ike.

5. Ihe ịma aka nka nka: Kedụ ka anyị si na izu oke?

N'agbanyeghị nnukwu nkwa ya, CRISPR-Cas9 ka na-eche ọtụtụ ihe ịma aka dị irè ihu na ngwa ụwa n'ezie. Dịka ọmụmaatụ, gbanyụọ - Mmetụta ebumnuche nwere ike webata ihe egwu nchekwa nwere ike ime. Usoro nnyefe na-achọkwa nkwalite ọzọ na arụmọrụ yana nchekwa. Na mgbakwunye, protein Cas9 n'onwe ya nwere ike ịkpalite nzaghachi mgbochi ọrịa n'ọnọdụ ụfọdụ. N'otu oge ahụ, a ga-eji nnukwu ịkpachara anya bịaruo nchegbu gbasara ụkpụrụ—karịsịa gburugburu ndezi nje nje.

Iji dozie okwu ndị a, ndị na-eme nchọpụta na-arụsi ọrụ ike na nhazi nke nkà na ụzụ. Ndị a gụnyere mmepe nke ụdị Cas9 dị elu - ikwesị ntụkwasị obi (dị ka HiFi Cas9) iji belata gbanyụọ - ọrụ ebumnuche, yana ọganihu nke atụmatụ ọhụrụ dị ka ndezi ntọala na ndezi isi, bụ nke chọrọ imeziwanye nkenke na njikwa njikwa mkpụrụ ndụ ihe nketa.

6. Echiche Ọdịnihu: “Sistemụ arụmọrụ” nke ọgwụgwọ cell

Ọ bụrụ na ọgwụ mgbochi mmadụ na-anọchi anya “ezigbo ntiwa,” mgbe ahụ CRISPR/Cas9 dị ka “ịdegharị koodu nke ndụ.” Ntuziaka ọdịnihu na-apụtawanye ìhè:

1. Gbanyụọ-ọgwụgwọ cell shelf
   Nnukwu mmepụta ụlọ ọrụ dị ukwuu iji belata ọnụ ahịa
2. Multiplex gene edezi
   Mgbanwe otu oge nke ọtụtụ mkpụrụ ndụ ihe nketa iji kwalite nrụpụta ọgwụgwọ
3. Ndozi vivo
   Ọgwụgwọ ozugbo n'ime ahụ mmadụ, na-ewepụ mkpa ọ dịex vivonhazi cell
4. AI + gene edezi
   Iji ọgụgụ isi arụ ọrụ iji bulie imewe gRNA wee belata - ihe egwu ebumnuche

Mmechi: Site na "Mkpa" gaa na usoro ịwa ahụ

Mpụta nke CRISPR/Cas9 nwere, na nke mbụ, nyere mmadụ ikike ịgbanwe mkpụrụ ndụ ihe nketa nke ọma. Na ngalaba nke CAR -T na ọgwụgwọ cell, ọ na-eme mgbanwe mgbanwe site na nanị "ịgwọ ọrịa" gaa na "ịrụghachi usoro ndu."

N'ile anya n'ihu n'afọ iri na-abịanụ, a na-atụ anya idezi mkpụrụ ndụ ihe nketa yana ọgwụgwọ cell ga-abụ isi injin nke ọgbọ na-esote biomedicine. CRISPR/Cas9 guzo na mmalite mmalite nke mgbanwe a.

Ntụaka

• [1] Doudna JA, Charpentier E. Oke ọhụrụ nke genome engineering na CRISPR-Cas9[J]. Sayensị, 2014, 346 (6213): 1258096.
• [2] Giuffrida L, Sek K, Henderson MA, et al. CRISPR/Cas9 mgbasa ozi ihichapụ nke adenosine A2A nnabata na-akwalite nrụpụta cell CAR T[J]. Nzikọrịta okike, 2021, 12(1): 3236.
• [3] Raguram A, Banskota S, Liu DR R. Ọgwụgwọna vivonnyefe nke ndị na-edezi mkpụrụ ndụ ihe nketa[J]. Cell, 2022, 185 (15): 2806-2827.