Revolutionising the CRISPR method

Researchers at ETH Zurich have refined the famous CRISPR-Cas method. Now, for the very first time, it is possible to modify dozens, if not hundreds, of genes in a cell simultaneously.

Genes and pro­teins in cells in­ter­act in many dif­fer­ent ways. Each dot rep­re­sents a gene; the lines are their in­ter­ac­tions. For the first time, the new method uses biotech­nol­ogy to in­flu­ence en­tire gene net­works in one sin­gle step. (Vi­su­al­iza­tions: ETH Zurich / Carlo Cosimo Campa)

Every­one’s talk­ing about CRISPR-Cas. This biotech­no­log­i­cal method of­fers a rel­a­tively quick and easy way to ma­nip­u­late sin­gle genes in cells, mean­ing they can be pre­cisely deleted, re­placed or mod­i­fied. Fur­ther­more, in re­cent years, re­searchers have also been us­ing tech­nolo­gies based on CRISPR-Cas to sys­tem­at­i­cally in­crease or de­crease the ac­tiv­ity of in­di­vid­ual genes. The cor­re­spond­ing meth­ods have be­come the world­wide stan­dard within a very short time, both in ba­sic bi­o­log­i­cal re­search and in ap­plied fields such as plant breed­ing.

To date, for the most part, re­searchers could mod­ify only one gene at a time us­ing the method. On oc­ca­sion, they man­aged two or three in one go; in one par­tic­u­lar case, they were able to edit seven genes si­mul­ta­ne­ously. Now, Pro­fes­sor Ran­dall Platt and his team at the De­part­ment of Biosys­tems Sci­ence and En­gi­neer­ing at ETH Zurich in Basel have de­vel­oped a process that – as they demon­strated in ex­per­i­ments – can mod­ify 25 tar­get sites within genes in a cell at once. As if that were not enough, this num­ber can be in­creased still fur­ther, to dozens or even hun­dreds of genes, as Platt points out. At any rate, the method of­fers enor­mous po­ten­tial for bio­med­ical re­search and biotech­nol­ogy. “Thanks to this new tool, we and other sci­en­tists can now achieve what we could only dream of do­ing in the past.”

Tar­geted, large-scale cell re­pro­gram­ming

Genes and pro­teins in cells in­ter­act in many dif­fer­ent ways. The re­sult­ing net­works com­pris­ing dozens of genes en­sure an or­gan­ism’s cel­lu­lar di­ver­sity. For ex­am­ple, they are re­spon­si­ble for dif­fer­en­ti­at­ing prog­en­i­tor cells to neu­ronal cells and im­mune cells. “Our method en­ables us, for the first time, to sys­tem­at­i­cally mod­ify en­tire gene net­works in a sin­gle step,” Platt says.

More­over, it paves the way for com­plex, large-scale cell pro­gram­ming. It can be used to in­crease the ac­tiv­ity of cer­tain genes, while re­duc­ing that of oth­ers. The tim­ing of this change in ac­tiv­ity can also be pre­cisely con­trolled.

This is of in­ter­est for ba­sic re­search, for ex­am­ple in in­ves­ti­gat­ing why var­i­ous types of cells be­have dif­fer­ently or for the study of com­plex ge­netic dis­or­ders. It will also prove use­ful for cell re­place­ment ther­apy, which in­volves re­plac­ing dam­aged with healthy cells. In this case, re­searchers can use the method to con­vert stem cells into dif­fer­en­ti­ated cells, such as neu­ronal cells or in­sulin-pro­duc­ing beta cells, or vice versa, to pro­duce stem cells from dif­fer­en­ti­ated skin cells.

The dual func­tion of the Cas en­zyme

The CRISPR-Cas method re­quires an en­zyme known as a Cas and a small RNA mol­e­cule. Its se­quence of nu­cle­obases serves as an “ad­dress la­bel”, di­rect­ing the en­zyme with ut­most pre­ci­sion to its des­ig­nated site of ac­tion on the chro­mo­somes. ETH sci­en­tists have cre­ated a plas­mid, or a cir­cu­lar DNA mol­e­cule, that stores the blue­print of the Cas en­zyme and nu­mer­ous RNA ad­dress mol­e­cules, arranged in se­quences: in other words, a longer ad­dress list. In their ex­per­i­ments, the re­searchers in­serted this plas­mid into hu­man cells, thereby demon­strat­ing that sev­eral genes can be mod­i­fied and reg­u­lated si­mul­ta­ne­ously.

For the new tech­nique, the sci­en­tists did not use the Cas9 en­zyme that has fea­tured in most CRISPR-Cas meth­ods to date, but the re­lated Cas12a en­zyme. Not only can it edit genes, it can also cut the long “RNA ad­dress list” into in­di­vid­ual “ad­dress la­bels” at the same time. Fur­ther­more, Cas12a can han­dle shorter RNA ad­dress mol­e­cules than Cas9. “The shorter these ad­dress­ing se­quences are, the more of them we can fit onto a plas­mid,” Platt says.

By:  Fabio Bergamin

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